Abstract
A high NH4+ load is known to inhibit bacterial methane oxidation. This is due to a competition between CH4 and NH3 for the active site of particulate methane monooxygenase (pMMO), which converts CH4 to CH3OH. Here, we combined global proteomics with amino acid profiling and nitrogen oxides measurements to elucidate the cellular acclimatization response of Methylocystis sp. strain SC2 to high NH4+ levels. Relative to 1 mM NH4+, a high (50 mM and 75 mM) NH4+ load under CH4-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 NH4+ 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 Km value for CH4 oxidation during the growth phase was indicative of increased pMMO-based oxidation of NH3 to toxic hydroxylamine. The detoxifying activity of hydroxlyamine oxidoreductase (HAO) led to a significant accumulation of NO2- and, upon decreasing O2 tension, N2O. Nitric oxide reductase and hybrid cluster proteins (Hcps) were the candidate enzymes for the production of N2O. In summary, strain SC2 has the capacity to precisely rebalance enzymes and osmolyte composition in response to increasing NH4+ 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 NH4+. IMPORTANCE In addition to reducing CH4 emissions from wetlands and landfills, the activity of alphaproteobacterial methane oxidizers of the genus Methylocystis contributes to the sink capacity of forest and grassland soils for atmospheric methane. The methane-oxidizing activity of Methylocystis spp. is, however, sensitive to high NH4+ concentrations. This is due to the competition of CH4 and NH3 for the active site of particulate methane monooxygenase, thereby resulting in the production of toxic hydroxylamine with an increasing NH4+ load. An understanding of the physiological and molecular response mechanisms of Methylocystis 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 Methylocystis sp. strain SC2 to an increasing NH4+ load.