sp. Strain SC2 Acclimatizes to Increasing NH Levels by a Precise Rebalancing of Enzymes and Osmolyte Composition.

A high NH load is known to inhibit bacterial methane oxidation. This is due to a competition between CH and NH for the active site of particulate methane monooxygenase (pMMO), which converts CH to CHOH. Here, we combined global proteomics with amino acid profiling and nitrogen oxides measurements to elucidate the cellular acclimatization response of sp. strain SC2 to high NH levels. Relative to 1 mM NH, a high (50 mM and 75 mM) NH load under CH-replete conditions significantly increased the lag phase duration required for proteome adjustment. The number of differentially regulated proteins was highly significantly correlated with an increasing NH load. The cellular responses to increasing ionic and osmotic stress involved a significant upregulation of stress-responsive proteins, the K "salt-in" strategy, the synthesis of compatible solutes (glutamate and proline), and the induction of the glutathione metabolism pathway. A significant increase in the apparent value for CH oxidation during the growth phase was indicative of increased pMMO-based oxidation of NH to toxic hydroxylamine. The detoxifying activity of hydroxlyamine oxidoreductase (HAO) led to a significant accumulation of NO and, upon decreasing O tension, NO. Nitric oxide reductase and hybrid cluster proteins (Hcps) were the candidate enzymes for the production of NO. In summary, strain SC2 has the capacity to precisely rebalance enzymes and osmolyte composition in response to increasing NH exposure, but the need to simultaneously combat both ionic-osmotic stress and the toxic effects of hydroxylamine may be the reason why its acclimatization capacity is limited to 75 mM NH. In addition to reducing CH emissions from wetlands and landfills, the activity of alphaproteobacterial methane oxidizers of the genus contributes to the sink capacity of forest and grassland soils for atmospheric methane. The methane-oxidizing activity of spp. is, however, sensitive to high NH concentrations. This is due to the competition of CH and NH for the active site of particulate methane monooxygenase, thereby resulting in the production of toxic hydroxylamine with an increasing NH load. An understanding of the physiological and molecular response mechanisms of spp. is therefore of great importance. Here, we combined global proteomics with amino acid profiling and NOx measurements to disentangle the cellular mechanisms underlying the acclimatization of sp. strain SC2 to an increasing NH load.