title: Impact of Osmotic Stressors on the Metabolic Activity of Methylocystis sp. Strain SC2
title: Wirkung osmotischer Stressoren auf die metabolische Aktivität von Methylocystis sp. Stamm SC2
titleType: AlternativeTitle
Creators: name: Guo, Kangli
Publisher: name: Philipps-Universität Marburg
Year: 2023
Subjects: Life sciences
Biowissenschaften, Biologie

description: Proteobacterial methane-oxidizing bacteria, or methanotrophs, have the unique ability to grow on methane as their sole source of carbon and energy. Among these, Methylocystis spp. belong to the family Methylocystaceae within the Alphaproteobacteria. Their key enzyme is the particulate methane monooxygenase (pMMO), which oxidizes methane to methanol. Methylocystis spp. are among the ecologically most relevant methanotroph populations in terrestrial environments and are widely distributed in diverse habitats. In consequence, Methylocystis spp. require a range of physiological capabilities that allow them to respond and acclimatize to fluctuations in abiotic and biotic factors in their native environment. However, to date there still exist major gaps in our knowledge of their metabolic potential, in particular with regard to their ability to acclimatize to environmental change and to cope with abiotic stress. In my first project, we used a recently developed proteome workflow to elucidate the cellular mechanisms underlying the acclimatization of Methylocystis sp. strain SC2 to high NH4+ load (added as NH4Cl). Relative to 1 mM NH4+, high (50 mM and 75 mM) NH4+ load under CH4-replete conditions significantly increased the lag phase duration required for proteome adjustment, while the addition of 100 mM NH4+ completely inhibited growth of strain SC2. The number of differentially regulated proteins was highly significantly correlated to the increase in NH4+ load. The cellular responses involved the significant upregulation of stress-responsive proteins, the K+ “salt-in” strategy, the synthesis of compatible solutes (glutamate and proline), and the glutathione metabolism. The apparent Km value for CH4 oxidation significantly increased with the NH4+ load. This observation was indicative of an increased pMMO-based oxidation of NH3 to toxic hydroxylamine. In consequence, the detoxifying activity of hydroxlyamine oxidoreductase (HAO) increased with the NH4+ concentration and led to a significant accumulation of NO2− and, with delay, N2O. Significant production of N2O occurred only after the oxygen concentration had dropped to low or unmeasurable levels. Thus, high NH4+ load had a dual effect on the activity of strain SC2, with one being general phenomenon of ionic-osmotic stress and the other being the competitive inhibition effect of NH3 on pMMO-based methane oxidation. Although strain SC2 precisely rebalanced enzymes and osmolyte composition in response to the increase in NH4+ load, 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+. Starting point of my second project was the knowledge that the growth of strain SC2 is completely inhibited at medium concentrations  1.5% NaCl. Sodium chloride is an important ionic-osmotic stressor in bulk and rhizosphere soils. We therefore tested various amino acids and other osmolytes for their potential to act as a compatible solute or osmoprotectant under otherwise inhibitory NaCl conditions. The addition of 10 mM asparagine to the growth medium had the greatest stress relief effect under severe salinity (1.5% NaCl), leading to a partial growth recovery of strain SC2. The analysis of the exo-metabolome revealed that asparagine was taken up quantitatively by strain SC2. This resulted in an intracellular concentration of 264 ± 57 mM asparagine. Under severe salinity (1.5% NaCl), the uptake of asparagine induced major proteome rearrangements related to the KEGG level 2 categories energy metabolism, amino acid metabolism, and cell growth and death. In particular, various proteins involved in cell division and peptidoglycan synthesis showed a positive expression response. The incorporation of asparagine-derived 13C-carbon into nearly all amino acids indicated that asparagine acted as a source for cell biomass under severe salinity (1.5% NaCl), with glutamate being a major hub between central carbon and amino acid pathways.