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author:("Kim, uni")
1.  Identification of the Major Expressed S-Layer and Cell Surface-Layer-Related Proteins in the Model Methanogenic Archaea: Methanosarcina barkeri Fusaro and Methanosarcina acetivorans C2A 
Archaea  2012;2012:873589.
Many archaeal cell envelopes contain a protein coat or sheath composed of one or more surface exposed proteins. These surface layer (S-layer) proteins contribute structural integrity and protect the lipid membrane from environmental challenges. To explore the species diversity of these layers in the Methanosarcinaceae, the major S-layer protein in Methanosarcina barkeri strain Fusaro was identified using proteomics. The Mbar_A1758 gene product was present in multiple forms with apparent sizes of 130, 120, and 100 kDa, consistent with post-translational modifications including signal peptide excision and protein glycosylation. A protein with features related to the surface layer proteins found in Methanosarcina acetivorans C2A and Methanosarcina mazei Goel was identified in the M. barkeri genome. These data reveal a distinct conserved protein signature with features and implied cell surface architecture in the Methanosarcinaceae that is absent in other archaea. Paralogous gene expression patterns in two Methanosarcina species revealed abundant expression of a single S-layer paralog in each strain. Respective promoter elements were identified and shown to be conserved in mRNA coding and upstream untranslated regions. Prior M. acetivorans genome annotations assigned S-layer or surface layer associated roles of eighty genes: however, of 68 examined none was significantly expressed relative to the experimentally determined S-layer gene.
PMCID: PMC3361143  PMID: 22666082
2.  S-layer Surface-Accessible and Concanavalin A Binding Proteins of Methanosarcina acetivorans and Methanosarcina mazei 
Journal of proteome research  2009;8(4):1972-1982.
The outermost cell envelope structure of many archaea and bacteria contains a proteinaceous lattice termed the surface layer or S-layer. It is typically composed of only one or two abundant, often post-translationally modified proteins that self-assemble to form the highly organized arrays. Surprisingly, over a hundred proteins were annotated to be S-layer components in the archaeal species Methanosarcina acetivorans C2A and Methanosarcina mazei Gö1, reflecting limitations of current predictions. An in vivo biotinylation methodology was devised to affinity tag surface-exposed proteins while overcoming unique challenges in working with these fragile organisms. Cells were adapted to growth under N2 fixing conditions, thus minimizing free amines reactive to the NHS-label, and high pH media compatible with the acylation chemistry was used. A 3-phase separation procedure was employed to isolate intact, labeled cells from lysed-cell derived proteins. Streptavidin affinity enrichment followed by stringent wash conditions removed non-specifically bound proteins. This methodology revealed S-layer proteins in M. acetivorans C2A and M. mazei Gö1 to be MA0829 and MM1976, respectively. Each was demonstrated to exist as multiple glycosylated forms using SDS-PAGE coupled with glycoprotein-specific staining, and by interaction with the lectin, Concanavalin A. A number of additional surface-exposed proteins and glycoproteins were identified and included all three subunits of the thermosome: the latter suggests that the chaperonin complex is both surface- and cytoplasmically-localized. This approach provides an alternative strategy to study surface proteins in the archaea.
PMCID: PMC2666069  PMID: 19228054
S-layer; surface proteins; Methanosarcina acetivorans; Methanosarcina mazei; biotinylation; mass spectrometry; glycoproteins; Concanavalin A
3.  Quantitative Proteomic and Microarray Analysis of the Archaeon Methanosarcina Acetivorans Grown with Acetate Versus Methanol* 
Journal of proteome research  2007;6(2):759-771.
Methanosarcina acetivorans strain C2A is an acetate- and methanol-utilizing methane-producing organism for which the genome, the largest yet sequenced among the Archaea, reveals extensive physiological diversity. LC linear ion trap-FTICR mass spectrometry was employed to analyze acetate- vs. methanol-grown cells metabolically labeled with 14N vs. 15N, respectively, to obtain quantitative protein abundance ratios. DNA microarray analyses of acetate- vs. methanol-grown cells was also performed to determine gene expression ratios. The combined approaches were highly complementary, extending the physiological understanding of growth and methanogenesis. Of the 1081 proteins detected, 255 were ≥ 3-fold differentially abundant. DNA microarray analysis revealed 410 genes that were ≥ 2.5-fold differentially expressed of 1972 genes with detected expression. The ratios of differentially abundant proteins were in good agreement with expression ratios of the encoding genes. Taken together, the results suggest several novel roles for electron transport components specific to acetate-grown cells, including two flavodoxins each specific for growth on acetate or methanol. Protein abundance ratios indicated that duplicate CO dehydrogenase/acetyl-CoA complexes function in the conversion of acetate to methane. Surprisingly, the protein abundance and gene expression ratios indicated a general stress response in acetate- vs. methanol-grown cells that included enzymes specific for polyphosphate accumulation and oxidative stress. The microarray analysis identified transcripts of several genes encoding regulatory proteins with identity to the PhoU, MarR, GlnK, and TetR families commonly found in the Bacteria domain. An analysis of neighboring genes suggested roles in controlling phosphate metabolism (PhoU), ammonia assimilation (GlnK), and molybdopterin cofactor biosynthesis (TetR). Finally, the proteomic and microarray results suggested roles for two-component regulatory systems specific for each growth substrate.
PMCID: PMC2577390  PMID: 17269732
Quantitative proteomics; metabolic labeling; microarray; methanogenesis; acetate; methanol
4.  Heat Shock Response of Archaeoglobus fulgidus†  
Journal of Bacteriology  2005;187(17):6046-6057.
The heat shock response of the hyperthermophilic archaeon Archaeoglobus fulgidus strain VC-16 was studied using whole-genome microarrays. On the basis of the resulting expression profiles, approximately 350 of the 2,410 open reading frames (ORFs) (ca. 14%) exhibited increased or decreased transcript abundance. These span a range of cell functions, including energy production, amino acid metabolism, and signal transduction, where the majority are uncharacterized. One ORF called AF1298 was identified that contains a putative helix-turn-helix DNA binding motif. The gene product, HSR1, was expressed and purified from Escherichia coli and was used to characterize specific DNA recognition regions upstream of two A. fulgidus genes, AF1298 and AF1971. The results indicate that AF1298 is autoregulated and is part of an operon with two downstream genes that encode a small heat shock protein, Hsp20, and cdc48, an AAA+ ATPase. The DNase I footprints using HSR1 suggest the presence of a cis-binding motif upstream of AF1298 consisting of CTAAC-N5-GTTAG. Since AF1298 is negatively regulated in response to heat shock and encodes a protein only distantly related to the N-terminal DNA binding domain of Phr of Pyrococcus furiosus, these results suggest that HSR1 and Phr may belong to an evolutionarily diverse protein family involved in heat shock regulation in hyperthermophilic and mesophilic Archaea organisms.
PMCID: PMC1196131  PMID: 16109946

Results 1-4 (4)