UCLA

Transcriptomics is often touted to be the silver bullet to determining the protein levels within a microorganism when comparing such proteins under two or more different biological conditions. However, designating the transcriptome as a surrogate for protein abundance can be misleading and impractical when determining the effect of environmental stresses on the phenotype of an organism. While the transcriptome is significant for identifying genes that are linked to, or act as co-regulators in the response to stressors, it is best to consider proteomic analyses, in addition to transcriptional profiling, as a complementary tool in order to determine the effect of different environmental factors on the fitness and phenotype of the microorganism in question. Methanosarcina mazei are mesophilic archaea microorganisms that grow under anaerobic conditions and produce methane via an energy-conservation process, known as methanogenesis, which is the terminal step in the degradation of organic matter during the carbon cycle. While the Methanosarcina are versatile in their ability to grow on numerous substrates, such as methanol and other compounds such as monomethylamine (MMA), dimethylamine (DMA), and trimethylamine (TMA), it is more thermodynamically favorable for the cells to grow on methanol (∆G’o = -106 kJ/ mol methane) than the methylamines (∆G’o = -77 kJ/ mol methane). Therefore, the lower free energy change in using the methylamines necessitates M. mazei cells to have an efficient energy-conserving system to deal with the thermodynamic limitations. Consequently absolute quantitation using a label-free proteomics strategy was applied to determine the intercelluar protein abundances of the Methanosarcina mazei proteome with different methylotrophic substrates (i.e., methanol, MMA, DMA, and TMA). Specifically, the aims of this project are to use quantitative proteomics to: (1) establish how M. mazei uses enzymes involved in methanogenesis to convert chemical energy into biomass; (2) determine how methanogenic process depends upon substrate type during the mid-log and stationary phases of growth; (3) determine whether different salt concentrations affect the glycosylation pattern of surface of the protein, and in turn methanogenesis; (4) understand the effect of methylotrophic substrate availability on a different methanogen, M. barkeri. At least 40% of all of the proteins were found to be methyltransferases and methylcoenzyme-M reductases in M. mazei cells grown under mid-log and stationary phase conditions. Furthermore, the protein abundances varied in a substrate-specific manner, indicating that the M. mazei cells may have evolved to be prepared for a potential carbon source switch.