[Show abstract][Hide abstract] ABSTRACT: 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 > or =3-fold differentially abundant. DNA microarray analysis revealed 410 genes that were > or =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.
Full-text · Article · Feb 2007 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: Methanosarcina acetivorans produces acetate, formate, and methane when cultured with CO as the growth substrate [Rother M, Metcalf WW (2004) Proc Natl Acad Sci USA 101:], which suggests novel features of CO metabolism. Here we present a genome-wide proteomic approach to identify and quantify proteins differentially abundant in response to growth on CO versus methanol or acetate. The results indicate that oxidation of CO to CO2 supplies electrons for reduction of CO2 to a methyl group by steps and enzymes of the pathway for CO2 reduction determined for other methane-producing species. However, proteomic and quantitative RT-PCR results suggest that reduction of the methyl group to methane involves novel methyltransferases and a coenzyme F420H2:heterodisulfide oxidoreductase system that generates a proton gradient for ATP synthesis not previously described for pathways reducing CO2 to methane. Biochemical assays support a role for the oxidoreductase, and transcriptional mapping identified an unusual operon structure encoding the oxidoreductase. The proteomic results further indicate that acetate is synthesized from the methyl group and CO by a reversal of initial steps in the pathway for conversion of acetate to methane that yields ATP by substrate level phosphorylation. The results indicate that M. acetivorans utilizes a pathway distinct from all known CO2 reduction pathways for methane formation that reflects an adaptation to the marine environment. Finally, the pathway supports the basis for a recently proposed primitive CO-dependent energy-conservation cycle that drove and directed the early evolution of life on Earth.
Full-text · Article · Dec 2006 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: A new algorithm (QN) for the (15)N /(14)N quantitation of relative protein abundances in complex proteomic samples is described. QN takes advantage of the high resolution, mass accuracy and throughput of the hybrid mass spectrometer LTQ-FT MS. Peptide quantitation is based on MS peak intensity (measured in the FT MS), while peptide identification is performed in the MS/MS mode (measured in the LTQ linear ion trap). Accuracy of the protein abundance is enhanced by a novel scoring procedure, allowing filtering of less reliable measurements of peptide abundances. The performance of QN is illustrated in the relative quantitative analysis of M. acetivorans C2A cultures grown with carbon monoxide vs methanol as substrate. Roughly 1,000 proteins were quantitated with an average CV of 9% for the protein abundance ratios. QN performs quantitation without manual intervention, does not require high processing power, and generates files compatible with the Guidelines for Proteomic Data Publication.
Full-text · Article · Sep 2006 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: A liquid chromatography-hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry approach was used
to determine the differential abundance of proteins in acetate-grown cells compared to that of proteins in methanol-grown
cells of the marine isolate Methanosarcina acetivorans metabolically labeled with 14N versus 15N. The 246 differentially abundant proteins in M. acetivorans were compared with the previously reported 240 differentially expressed genes of the freshwater isolate Methanosarcina mazei determined by transcriptional profiling of acetate-grown cells compared to methanol-grown cells. Profound differences were
revealed for proteins involved in electron transport and energy conservation. Compared to methanol-grown cells, acetate-grown
M. acetivorans synthesized greater amounts of subunits encoded in an eight-gene transcriptional unit homologous to operons encoding the
ion-translocating Rnf electron transport complex previously characterized from the Bacteria domain. Combined with sequence and physiological analyses, these results suggest that M. acetivorans replaces the H2-evolving Ech hydrogenase complex of freshwater Methanosarcina species with the Rnf complex, which generates a transmembrane ion gradient for ATP synthesis. Compared to methanol-grown
cells, acetate-grown M. acetivorans synthesized a greater abundance of proteins encoded in a seven-gene transcriptional unit annotated for the Mrp complex previously
reported to function as a sodium/proton antiporter in the Bacteria domain. The differences reported here between M. acetivorans and M. mazei can be attributed to an adaptation of M. acetivorans to the marine environment.
Full-text · Article · Feb 2006 · Journal of Bacteriology
[Show abstract][Hide abstract] ABSTRACT: A fundamental goal ingenomics is the discovery of genetic variation that contributes to disease states or to differential drug responses. Single nucleotide polymorphism (SNP) detection has been the focus of much attention in the study of genetic variation over the last decade. These SNPs typically occur at a frequency greater than 1% in the human genome. Recently, low-frequency alleles are also being increasingly recognized as critical to obtain an improved understanding of the correlation between genetic variation and disease. Although many methods have been reported for the discovery and scoringof SNPs, sensitive, automated, and cost-effective methods and platforms for the discovery of low-frequency alleles are not yet readily available. We describe here an automated multicapillary instrument for high-throughput detection of low-frequency alleles from pooled samples using constant denaturant capillary electrophoresis. The instrument features high optical sensitivity (1 x 10(-12) M fluorescein detection limit), precise and stable temperature control (+/- 0.01degrees C), and automation for sample delivery, injection, matrix replacement, and fraction collection. The capillary array is divided into six groups of four capillaries, each of which can be independently set at any temperature ranging from room temperature to 90 degrees C. The key performance characteristics of the instrument are reported.
No preview · Article · Aug 2005 · Journal of Separation Science
[Show abstract][Hide abstract] ABSTRACT: Methanosarcina acetivorans is an archaeon isolated from marine sediments which utilizes a diversity of substrates for growth and methanogenesis. Part I of a two-part investigation has profiled proteins of this microorganism cultured with both methanol and acetate as growth substrates, utilizing two-dimensional gel electrophoresis and MALDI-TOF-TOF mass spectrometry. In this report, Part II, the analyses were extended to identify 34 proteins found to be present in different amounts between methanol- and acetate-grown M. acetivorans. Among these proteins are enzymes which function in pathways for methanogenesis from either acetate or methanol. Several of the 34 proteins were determined to have redundant functions based on annotations of the genomic sequence. Enzymes which function in ATP synthesis and steps common to both methanogenic pathways were elevated in acetate- versus methanol-grown cells, whereas enzymes that have a more general function in protein synthesis were in greater amounts in methanol- compared to acetate-grown cells. Several group I chaperonins were present in greater amounts in methanol- versus acetate-grown cells, whereas lower amounts of several stress related proteins were found in methanol- versus acetate-grown cells. The potential physiological basis for these novel patterns of protein synthesis are discussed.
Full-text · Article · Feb 2005 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: Methanosarcina acetivorans is representative of the genus that is distinguished from all other methane-producing genera by extensive metabolic diversity predicted from the large genome. In Part I of this study, two-dimensional gel electrophoresis and MALDI-TOF-TOF mass spectrometry was used to investigate the proteome of methanol- or acetate-grown M. acetivorans, with the goal of an initial characterization of the diversity of the proteins synthesized. A total of 412 proteins were identified, representing nearly 10% of the ORFs, with nearly 30% conserved hypothetical or hypothetical. Of the 412 proteins, 188 were found in both acetate- and methanol-grown cells, 122 were detected only in acetate-grown cells, and 102 only in methanol-grown cells. The results revealed the expression of a remarkable number of redundant genes which encode enzymes involved in the pathways for methanogenesis from methanol or acetate, suggesting an important role for the unusually high percentage of redundant genes in Methanosarcina species. Evidence was obtained for synthesis of a sodium-transporting oxidoreductase in acetate-grown cells, with the potential to function in energy conservation. Several transcriptional regulatory proteins were identified that also function in the Bacteria domain, raising questions regarding their interaction with the Archaea/Eucarya-type basal transcription apparatus. In addition, a significant number of proteins involved in protein folding were shown to be synthesized in methanol- and acetate-grown cells. These studies provide the first examination of the protein diversity of M. acetivorans.
Full-text · Article · Feb 2005 · Journal of Proteome Research