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Acclimatization of Methylocystis to inhibitory NH4+ levels by multiple proteomic and metabolic response mechanisms

ABSTRACT: High NH4+ load is known to competitively inhibit bacterial methane oxidation. This is due to a competition between CH4 and NH4+/NH3 for the active site of particulate methane monooxygenase (pMMO), which converts CH4 to CH3OH. Here, we combined growth experiments with global proteomics to elucidate the capability of the methanotroph Methylocystis sp. strain SC2 in acclimatizing to increased NH4+ levels. Our experimental approach also involved amino acid profiling and measurement of NOx compounds. Relative to 1 mM NH4+, high (50 mM and 75 mM) NH4+ load under CH4 replete conditions significantly increased lag phase duration required for proteome adjustment. The proteomic and metabolic responses to increasing ionic and osmotic stress involved significant upregulation of stress-responsive proteins, K+ “salt in” strategy, synthesis of compatible solutes (glutamate and proline), and 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 NH4+/NH3 to toxic hydroxylamine. The detoxifying activity of hydroxlyamine oxidoreductase (HAO) led to a significant accumulation of NO2- and, upon decreasing O2 tension, N2O. Putative free intermediate of HAO activity was NO, with NO reductase and hybrid cluster proteins (Hcps) being the candidate enzymes for the reduction of NO to N2O. In summary, strain SC2 has the capacity to precisely rebalance enzymes and osmolyte composition, 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+.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Methylocystis Sp. Sc2

TISSUE(S): Cell Culture

SUBMITTER: Timo Glatter

LAB HEAD: Timo Glatter

PROVIDER: PXD032347 | Pride | 2022-09-19

REPOSITORIES: Pride

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Project description:Oxygen deficient zones (ODZs) are major sites of net natural oceanic nitrous oxide (N2O) production and emissions. In order to understand changes in the magnitude of N2O production in response to global change, knowledge on the individual contributions of the major microbial pathways (nitrification and denitrification) to N2O production and their regulation is needed. In the ODZ of the coastal area off Peru, the sensitivity of N2O production to oxygen and organic matter was investigated using 15N-tracer experiments in combination with qPCR and microarray analysis of total and active functional genes targeting archaeal amoA and nirS as marker genes for nitrification and denitrification, respectively. Denitrification was responsible for the highest N2O production with mean 8.7 nmol L-1 d-1 but up to 118 Â± 27.8 nmol L-1 d-1 just below the oxic-anoxic interface. Highest N2O production from AO of 0.16 Â± 0.003 nmol L-1 d-1 occurred in the upper oxycline at O2 concentrations of 10 - 30 Âµmol L-1 which coincided with highest archaeal amoA transcripts/genes. Oxygen responses of N2O production varied with substrate, but production and yields were generally highest below 10 Âµmol L-1 O2. Particulate organic matter additions increased N2O production by denitrification up to 5-fold suggesting increased N2O production during times of high particulate organic matter export. High N2O yields from ammonium oxidation of 2.1% were measured, but the overall contribution to N2O production stays an order of magnitude behind denitrification as an N2O source. Hence, these findings show that denitrification is the most important N2O production process in low oxygen conditions fueled by organic carbon supply which implies a positive feedback of the total oceanic N2O sources in response to increasing oceanic deoxygenation. [SUBMITTER_CITATION]: Frey, C., Bange, H. W., Achterberg, E. P., Jayakumar, A., Löscher, C. R., Arévalo-Martínez, D. L., León-Palmero, E., Sun, M., Sun, X., Xie, R. C., Oleynik, S., and Ward, B. B.: Regulation of nitrous oxide production in low-oxygen waters off the coast of Peru, Biogeosciences, 17, 2263-2287

			Action	DRS
	KLG-3-10-01.msf	Msf
	KLG-3-10.raw	Raw
	KLG-3-11-01.msf	Msf
	KLG-3-11.raw	Raw
	KLG-3-12-01.msf	Msf

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Acclimatization of Methylocystis to inhibitory NH4+ levels by multiple proteomic and metabolic response mechanisms

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