Dokument

Summary:
Microbial methane oxidation is one of the fundamental processes in global methane cycle. Methane-oxidizing bacteria, or methanotrophs, are the major biological sink for the methane produced from anthropogenic and natural sources. Our model organism, Methylocystis sp. strain SC2, is one of the best-studied representatives of alphaproteobacterial (type IIa) methanotrophs. Proteobacterial methanotrophs possess a unique cell architecture characterized by intracytoplasmic membranes (ICMs). The cellular amount of the ICMs is increasing with methanotrophic activity. The presence of ICMs makes molecular biology approaches, but in particular global proteomics, highly challenging. In this study, we therefore aimed to develop an efficient proteomics workflow for strain SC2 and to apply this state-of-the-art tool for investigation of the strain SC2 response to environmental factors. To successfully develop the proteomics workflow, we particularly focused on an efficient solubilization and digestion of the integral membrane proteins of strain SC2 for further downstream analysis. We introduced the so-called crude-MS proteomics workflow, upon assessing and optimizing all the major steps in the proteomics workflow, including cell lysis, protein solubilization, and protein digestion. Our new SC2 proteomics workflow greatly increased not only the protein quantification accuracy (mean coefficient of variation 3.2 %) but also the proteome coverage to 62%, with up to 10-fold increase in the detection intensity of membrane-associated proteins. Previous studies have shown that the LysC/trypsin tandem digestion resulted in higher coverage of fully cleaved tryptic peptides than a trypsin-only digestion. Therefore, the development of our optimized proteomics workflow involved the application of the LysC/trypsin tandem digestion in detergent environment to increase the SC2 proteome coverage. Prior to publication of our crude-MS approach, all systematic assessments of LysC/trypsin proteolysis were conducted in chaotropic environments, like urea. As a spin-off, we therefore initiated a follow-up study to compare the efficiency of the LysC/trypsin tandem digestion in detergent environments (e.g., SDC, SLS) relative to chaotropic environments. The study revealed that the LysC/trypsin tandem digestion could be efficiently carried out not only in chaotropic environments but also in MS-compatible detergent environments. In fact, the LysC/trypsin tandem digestion in both environments resulted in a higher coverage of fully cleaved peptides than the trypsin-only digestion. After successful development of the crude-MS proteomics workflow, we used this high-throughput method to assess the molecular response of strain SC2 to the availability of hydrogen as a potentially alternative energy source. Starting point of this research was the knowledge that strain SC2 and other Methylocystis spp. possess the genetic potential to produce various hydrogenases. In fact, the addition of 2% hydrogen to the headspace atmosphere led, under limiting concentrations of methane and oxygen, to the complete hydrogen consumption by strain SC2. Concurrently, the SC2 biomass yield was significantly increased, while the methane consumption rate was significantly decreased. Global proteome analyses revealed that the addition of hydrogen induced an increase in the production of Group 1d and Group 2b [NiFe]-hydrogenases, and hydrogenase accessory proteins. Notably, the upregulation of the Group 1d, 2b [NiFe]-hydrogenases was concomitantly linked to a reconstruction of the energy metabolism in strain SC2. In another project, genome-scale metabolic modeling and growth experiments were applied to show that strain SC2 has the capacity to utilize acetate through the glyoxylate assimilation pathway. In addition, the study revealed that in type II methanotrophs, energy demand for methane oxidation is covered by complex I of the electron transport chain. In summary, our research demonstrates how to experimentally link the metabolic potential of Methylocystis sp. strain SC2 with the underlying proteome complexity. Thus, the newly developed highly reproducible SC2 proteomics workflow represents a high-throughput method that makes it possible to achieve in future research an understanding of the molecular adaptation mechanisms of strain SC2 to environmental change.

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