Project description:Using recent developments in sample preparation strategies and improvements in mass spectrometry (MS), an optimized procedure was developed to characterize the proteome of Methylocystis sp. strain SC2, a type II methanotroph. It represents one of the ecologically important groups of methane-oxidizing bacteria. The major challenge for developing an efficient analytical proteomics workflow for methanotrophic bacteria is the high amount of membrane-associated proteins that need to be efficiently solubilized and digested for downstream analysis. Therefore, each step of the workflow, including cell lysis, protein solubilization and digestion, and MS peptide quantification, was assessed and optimized. Our novel crude-lysate-MS approach proved to increase protein quantification accuracy and the proteome coverage of strain SC2. It captured 62% of predicted SC2 proteome, with 10-fold increase in membrane-associated proteins relative to less effective conditions. Use of crude cell lysate for downstream analysis showed not only to be highly efficient for strain SC2 but also for other members of the Methylocystaceae family. To validate the efficiency of our newly developed workflow, we analyzed the SC2 proteome under two contrasting nitrogen conditions, with a focus on the differential expression of proteins involved in methane and nitrogen metabolisms.
Project description:The objective of this study was to assess whether Methylocystis sp. strain SC2, as a representative for Methylocystis spp., can utilize hydrogen to optimize the biomass yield by mixed utilization of CH4 and H2, rather than CH4 as the sole source of energy. Thus, we aimed to show that, in the presence of H2, CH4 will primarily be used for synthesis of cell carbon and increased biomass/protein yield. In particular, we intended to explore those CH4/O2 ratios, which maximize the effect of hydrogen addition on the biomass yield and proteome reconstruction of strain SC2. To achieve our goals, we combined hydrogen-based growth experiments with our recently optimized proteomics workflow.
Project description:Here we present the assembled genome of the facultative methanotroph, Methylocystis strain SB2, along with assessment of its transcriptome when grown on methane vs. ethanol. As expected, transcriptomic analyses indicate methane is converted to carbon dioxide via the canonical methane oxidation pathway for energy generation, and that carbon is assimilated at the level of formaldehyde via the serine cycle. When grown on ethanol, it appears this strain converts ethanol to acetyl-CoA and then utilizes the TCA cycle for energy generation and the ethylmalonyl CoA pathway for the production of biomass.
Project description:Here we present the assembled genome of the facultative methanotroph, Methylocystis strain SB2, along with assessment of its transcriptome when grown on methane vs. ethanol. As expected, transcriptomic analyses indicate methane is converted to carbon dioxide via the canonical methane oxidation pathway for energy generation, and that carbon is assimilated at the level of formaldehyde via the serine cycle. When grown on ethanol, it appears this strain converts ethanol to acetyl-CoA and then utilizes the TCA cycle for energy generation and the ethylmalonyl CoA pathway for the production of biomass. All cultures were grown in triplicates for subsequent DNA and RNA extraction as well as for subsequent sequencing using Illumina. Transcriptomic analysis results presented in this Series.
Project description: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+.
