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Functional analysis of the three TATA binding protein homologs in Methanosarcina acetivorans.

ABSTRACT: 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. Deltatbp3 and Deltatbp2 strains exhibited an extended lag phase compared with the wild type, although the lag phase for the Deltatbp2 strain was less pronounced when this strain was transitioning from growth on methylotrophic substrates to growth on acetate. Acetate-adapted Deltatbp3 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 Deltatbp2 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 Deltatbp2 and Deltatbp3 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.

SUBMITTER: Reichlen MJ

PROVIDER: S-EPMC2832540 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Functional analysis of the three TATA binding protein homologs in Methanosarcina acetivorans.

Reichlen Matthew J MJ Murakami Katsuhiko S KS Ferry James G JG

Journal of bacteriology 20100115 6

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 abundan ...[more]

PMID: 20081030

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Project description:BackgroundAcetate is the major source of methane in nature. The majority of investigations have focused on acetotrophic methanogens for which energy-conserving electron transport is dependent on the production and consumption of H₂ as an intermediate, although the great majority of acetotrophs are unable to metabolize H₂. The presence of cytochrome c and a complex (Ma-Rnf) homologous to the Rnf (Rhodobacter nitrogen fixation) complexes distributed in the domain Bacteria distinguishes non-H₂-utilizing Methanosarcina acetivorans from H₂-utilizing species suggesting fundamentally different electron transport pathways. Thus, the membrane-bound electron transport chain of acetate-grown M. acetivorans was investigated to advance a more complete understanding of acetotrophic methanogens.ResultsA component of the CO dehydrogenase/acetyl-CoA synthase (CdhAE) was partially purified and shown to reduce a ferredoxin purified using an assay coupling reduction of the ferredoxin to oxidation of CdhAE. Mass spectrometry analysis of the ferredoxin identified the encoding gene among annotations for nine ferredoxins encoded in the genome. Reduction of purified membranes from acetate-grown cells with ferredoxin lead to reduction of membrane-associated multi-heme cytochrome c that was re-oxidized by the addition of either the heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB) or 2-hydoxyphenazine, the soluble analog of methanophenazine (MP). Reduced 2-hydoxyphenazine was re-oxidized by membranes that was dependent on addition of CoM-S-S-CoB. A genomic analysis of Methanosarcina thermophila, a non-H2-utilizing acetotrophic methanogen, identified genes homologous to cytochrome c and the Ma-Rnf complex of M. acetivorans.ConclusionsThe results support roles for ferredoxin, cytochrome c and MP in the energy-conserving electron transport pathway of non-H₂-utilizing acetotrophic methanogens. This is the first report of involvement of a cytochrome c in acetotrophic methanogenesis. The results suggest that diverse acetotrophic Methanosarcina species have evolved diverse membrane-bound electron transport pathways leading from ferredoxin and culminating with MP donating electrons to the heterodisulfide reductase (HdrDE) for reduction of CoM-S-S-CoB.

Project description:Conserved genes essential to sulfur assimilation and trafficking in aerobic organisms are missing in many methanogens, most of which inhabit highly sulfidic, anaerobic environmental niches. This suggests that methanogens possess distinct pathways for the synthesis of key metabolites and intermediates, including cysteine, homocysteine, and protein persulfide groups. Prior work identified a novel tRNA-dependent two-step pathway for cysteine biosynthesis and a new metabolic transformation by which sulfur is inserted into aspartate semialdehyde to produce homocysteine. Homocysteine biosynthesis requires two of the three proteins previously identified in our laboratory by a comprehensive bioinformatics approach. Here, we show that the third protein identified in silico, the ApbE-like protein COG2122, facilitates sulfide assimilation in Methanosarcina acetivorans Knockout strains lacking the gene encoding COG2122 are severely impaired for growth when sulfide is provided as the sole sulfur source. However, rapid growth is recovered upon supplementation with cysteine, homocysteine, or cystathionine, suggesting that COG2122 is required for efficient biosynthesis of both cysteine and homocysteine. Deletion of the gene encoding COG2122 does not influence the extent of sulfur modifications in tRNA or the prevalence of iron-sulfur clusters, indicating that the function of COG2122 could be limited to sulfide assimilation for cysteine and homocysteine biosynthesis alone.We have found that the conserved M. acetivorans ma1715 gene, which encodes an ApbE-like protein, is required for optimal growth with sulfide as the sole sulfur source and supports both cysteine and homocysteine biosynthesis in vivo Together with related functional-genomics studies in methanogens, these findings make a key contribution to elucidating the novel pathways of sulfide assimilation and sulfur trafficking in anaerobic microorganisms that existed before the advent of oxygenic photosynthesis. The data suggest that the MA1715 protein is particularly important to sustaining robust physiological function when ambient sulfide concentrations are low. Phylogenetic analysis shows that MA1715 and other recently discovered methanogen sulfur-trafficking proteins share an evolutionary history with enzymes in the methanogenesis pathway. The newly characterized genes thus likely formed an essential part of the core metabolic machinery of the ancestral euryarchaeote.

Dataset Information

Functional analysis of the three TATA binding protein homologs in Methanosarcina acetivorans.

Publications

Functional analysis of the three TATA binding protein homologs in Methanosarcina acetivorans.

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