This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The archaea Methanosarcina acetivorans (M. acetivoranas) and Methanosarcina mazei (M. mazei), are extremely versatile anaerobe methanogens, capable of producing methane from three different methanogenesis pathways and nine different substrates, including acetate. As methane producers, they play a pivotal role in the global carbon cycle;they are major emitters of this greenhouse gas and thus could be exploited as generators of an alternative energy source. The Surface Layer (S-layer) of many prokaryotes and archaea, including the archaea M. mazei and M. acetivorans, is the outermost cell envelope structure formed by a highly organized proteinaceous 2-D crystalline array that self assembles, covering the entire cell surface. It is composed of one or two glycoprotein(s) with molecular weights (MWs) ranging from 40-200 kDa whose glycan chains protrude from the cell surface. This boundary layer is thought to play a critical role in the organisms'interactions with the external environment, nutrient uptake, cell excretion, signaling and surface interactions. For most archaea which lack a discrete cell wall, the S-layer acts as a protective coat and may determine and maintain cell shape. The Methanosarcinae are unique among these species in that they develop as multicellular bundles. In the laboratory, they can be grown as single cells or as multicellular packets, depending on the osmolarity of the medium. When grown at low osmolarity (freshwater medium), they form large multicellular structures encapsulated in a communal cyst;at high osmolarity (marine medium), they grow as single cells, each surrounded by an S-layer. Extensive bioinformatic efforts have tried to predict the cell envelope components and the S-layer protein(s) for M. mazei and M. acetivorans. Based on sequence homology and domain homology to other microbes S-layers, hundreds of Methanosarcinae genes have been annotated as S-layer proteins;these results showed the need for experimental measurements. In the first phase of Ms. Francoleon's PhD research, she focused on elucidation and characterization of the S-layer and surface proteins of M. mazei and M. acetivorans. The second phase of her PhD research further characterized the glycosylation of the S-layer protein (MM1976) and the surface proteins of M. mazei. The PhD research of Ms.Francoleon thus identified, for the first time, the surface proteins of the Archaea species, established that glycosylation is present as a post-translational modification and determined important characteristics of the S-layer glycoprotein glycans. However, largely because the UCLA laboratory has not previously engaged in carbohydrate structural determinations, it has not been possible for her to fully determine the glycan structures. Execution of her PhD project stimulated Ms. Francoleon's interest in glycobiology and determination to undertake post-doctoral training at a place where she can gain state-of-the-art experience in glycan analysis and then make her own contributions to the further development of the field. During her postdoctoral training, she is working to determine the structures and locations of the glycans, using methods developed at the Resource.
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