Quantitative Proteomic and Microarray Analysis of the Archaeon Methanosarcina acetivorans Grown with Acetate versus Methanol

Li, Lingyun; Li, Qingbo; Rohlin, Lars; Kim, Un Mi; Salmon, Kirsty; Rejtar, Tomáš; Gunsalus, Robert P.; Karger, Barry L.; Ferry, James G.

doi:10.1021/pr060383l

Cited by 94 publications

(130 citation statements)

References 71 publications

Supporting

Mentioning

118

Contrasting

Order By: Relevance

“…The Methanosarcina species are the most metabolically diverse species in the methane-producing Archaea (13,14), and the global gene expression profiles for various Methanosarcina species differ substantially during growth with different substrates (11,20,(26)(27)(28)(29)(30). However, how the pathways are regulated is largely unknown.…”

Section: Discussionmentioning

confidence: 99%

Functional Analysis of the Three TATA Binding Protein Homologs in Methanosarcina acetivorans

2010

Self Cite

View full text Add to dashboard Cite

The roles of three TATA binding protein (TBP) homologs (TBP1, TBP2, and TBP3) in the archaeon Methanosarcina acetivorans were investigated by using genetic and molecular approaches. Although tbp2 and tbp3 deletion mutants were readily obtained, a tbp1 mutant was not obtained, and the growth of a conditional tbp1 expression strain was tetracycline dependent, indicating that TBP1 is essential. Transcripts of tbp1 were 20-fold more abundant than transcripts of tbp2 and 100-to 200-fold more abundant than transcripts of tbp3, suggesting that TBP1 is the primary TBP utilized during growth. Accordingly, tbp1 is strictly conserved in the genomes of Methanosarcina species. ⌬tbp3 and ⌬tbp2 strains exhibited an extended lag phase compared with the wild type, although the lag phase for the ⌬tbp2 strain was less pronounced when this strain was transitioning from growth on methylotrophic substrates to growth on acetate. Acetate-adapted ⌬tbp3 cells exhibited growth rates, final growth yields, and lag times that were significantly reduced compared with those of the wild type when the organisms were cultured with growth-limiting concentrations of acetate, and the acetate-adapted ⌬tbp2 strain exhibited a final growth yield that was reduced compared with that of the wild type when the organisms were cultured with growth-limiting acetate concentrations. DNA microarray analyses identified 92 and 77 genes with altered transcription in the ⌬tbp2 and ⌬tbp3 strains, respectively, which is consistent with a role for TBP2 and TBP3 in optimizing gene expression. Together, the results suggest that TBP2 and TBP3 are required for efficient growth under conditions similar to the conditions in the native environment of M. acetivorans.The basal transcription machinery of the Archaea resembles that of the RNA polymerase II system in the Eukarya domain that includes two essential general transcription factors, TATA binding protein (TBP) and transcription factor B (TFB) (3-5, 16, 25, 42). In order to establish promoter-directed transcription, TBP binds a TATA box located ϳ25 bp upstream of the transcription start site. The binding of TBP to the promoter allows TFB to bind at sites both upstream and downstream of the TATA box. TFB then recruits RNA polymerase to the promoter to establish the preinitiation complex, which is followed by transcription (2).Genes encoding multiple homologs of TBP and/or TFB have been identified in the genomes of several species in the domain Archaea. It has been proposed that the function of the homologs is to direct gene-specific transcription, analogous to the function of alternative factors specific to the domain Bacteria (1). Experimental evidence that supports this hypothesis has been reported for the archaeon Halobacterium sp. NRC-1, which contains six tbp genes and seven tfb genes (9, 12). A comprehensive systems approach provided evidence suggesting that there is global gene regulation by specific TFB-TBP pairs (12). In another study, two different mutants in which there was deletion of either the tbpD or tfbA h...

show abstract

Section: Discussionmentioning

confidence: 99%

Functional Analysis of the Three TATA Binding Protein Homologs in Methanosarcina acetivorans

2010

Self Cite

View full text Add to dashboard Cite

show abstract

“…In metabolic labeling, dividing cells incorporate the label into their entire proteome. Of the two most common forms, 15 N labeling is usually used for microorganisms or small metazoan (38,39) whereas stable isotope labeling in cell culture (SILAC) is mostly used for mammalian cells (40,41). SILAC labels one or two specific amino acids, making peptide pairs easy to identify by virtue of their known mass differences.…”

Section: Accuracy In Protein Quantitation: the End Of One-size-fits-allmentioning

confidence: 99%

Precision proteomics: The case for high resolution and high mass accuracy

Mann

Kelleher

2008

Proc. Natl. Acad. Sci. U.S.A.

406

354

View full text Add to dashboard Cite

Proteomics has progressed radically in the last 5 years and is now on par with most genomic technologies in throughput and comprehensiveness. Analyzing peptide mixtures by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS) has emerged as the main technology for in-depth proteome analysis whereas two-dimensional gel electrophoresis, low-resolution MALDI, and protein arrays are playing niche roles. MS-based proteomics is rapidly becoming quantitative through both label-free and stable isotope labeling technologies. The latest generation of mass spectrometers combines extremely high resolving power, mass accuracy, and very high sequencing speed in routine proteomic applications. Peptide fragmentation is mostly performed in lowresolution but very sensitive and fast linear ion traps. However, alternative fragmentation methods and high-resolution fragment analysis are becoming much more practical. Recent advances in computational proteomics are removing the data analysis bottleneck. Thus, in a few specialized laboratories, ''precision proteomics'' can now identify and quantify almost all fragmented peptide peaks. Huge challenges and opportunities remain in technology development for proteomics; thus, this is not ''the beginning of the end'' but surely ''the end of the beginning.''

show abstract

“…In contrast to M. barkeri, Methanosarcina acetivorans lacks the Ech hydrogenase (11). It can nevertheless grow on acetate, which is why another complex present in this organism, the Rnf complex, is thought to be involved in the aceticlastic pathway of methanogenesis as an acceptor for Fd red (8,10,17). The Methanosarcina mazei genome, however, contains genes coding for the Ech hydrogenase, but this species lacks the Rnf complex (5).…”

mentioning

confidence: 99%

Function of Ech Hydrogenase in Ferredoxin-Dependent, Membrane-Bound Electron Transport in Methanosarcina mazei

et al. 2010

View full text Add to dashboard Cite

Reduced ferredoxin is an intermediate in the methylotrophic and aceticlastic pathway of methanogenesis and donates electrons to membrane-integral proteins, which transfer electrons to the heterodisulfide reductase. A ferredoxin interaction has been observed previously for the Ech hydrogenase. Here we present a detailed analysis of a Methanosarcina mazei ⌬ech mutant which shows decreased ferredoxin-dependent membranebound electron transport activity, a lower growth rate, and faster substrate consumption. Evidence is presented that a second protein whose identity is unknown oxidizes reduced ferredoxin, indicating an involvement in methanogenesis from methylated C 1 compounds.The aceticlastic pathway of methanogenesis creates approximately 70% (10) of the biologically produced methane and is of great ecological importance, as methane is a potent greenhouse gas. Organisms using this pathway to convert acetate to methane belong exclusively to the genera Methanosarcina and Methanosaeta. The two carbon atoms of acetate have different fates in the pathway. The methyl moiety is converted to methane, whereas the carbonyl moiety is further oxidized to CO 2 and the electrons derived from this oxidation step are used to reduce ferredoxin (Fd) (6). During methanogenesis from methylated C 1 compounds (methanol and methylamines), onequarter of the methyl groups are oxidized to obtain electrons for the reduction of heterodisulfide (27). A key enzyme in the oxidative part of methylotrophic methanogenesis is the formylmethanofuran dehydrogenase, which oxidizes the intermediate formylmethanofuran to CO 2 (7). The electrons are transferred to Fd. It has been suggested that reduced ferredoxin (Fd red ) donates electrons to the respiratory chain with the heterodisulfide (coenzyme M [CoM]-S-S-CoB) as the terminal electron acceptor and that the reaction is catalyzed by the Fd red :CoM-S-S-CoB oxidoreductase system (7, 24). The direct membranebound electron acceptor for Fd red is still a matter of debate; for the Ech hydrogenase, a reduced ferredoxin-accepting, H 2 -evolving activity has been observed for Methanosarcina barkeri (20), which implies that the H 2 :CoM-S-S-CoB oxidoreductase system is involved in electron transport (13). Direct electron flow from the Ech hydrogenase to the heterodisulfide reductase has not been shown to date (20,21). In contrast to M. barkeri, Methanosarcina acetivorans lacks the Ech hydrogenase (11). It can nevertheless grow on acetate, which is why another complex present in this organism, the Rnf complex, is thought to be involved in the aceticlastic pathway of methanogenesis as an acceptor for Fd red (8,10,17). The Methanosarcina mazei genome, however, contains genes coding for the Ech hydrogenase, but this species lacks the Rnf complex (5).To investigate whether the Ech hydrogenase is the only means by which M. mazei channels electrons from Fd red into the respiratory chain, a mutant lacking the Ech hydrogenase (M. mazei ⌬ech mutant) was constructed. Electron transport experiments using Fd red as the el...

show abstract

Quantitative Proteomic and Microarray Analysis of the Archaeon Methanosarcina acetivorans Grown with Acetate versus Methanol

Cited by 94 publications

References 71 publications

Functional Analysis of the Three TATA Binding Protein Homologs in Methanosarcina acetivorans

Functional Analysis of the Three TATA Binding Protein Homologs in Methanosarcina acetivorans

Precision proteomics: The case for high resolution and high mass accuracy

Function of Ech Hydrogenase in Ferredoxin-Dependent, Membrane-Bound Electron Transport in Methanosarcina mazei

Contact Info

Product

Resources

About