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ERIC Number: ED526003
Record Type: Non-Journal
Publication Date: 2009
Pages: 164
Abstractor: As Provided
Reference Count: 0
ISBN: ISBN-978-1-1095-8481-3
Analysis of the MT1/MT2 Systems Involved in the Metabolism of One-Carbon Compounds in "Methanosarcina acetivorans" C2A
Opulencia, Rina Bagsic
ProQuest LLC, Ph.D. Dissertation, University of Illinois at Urbana-Champaign
Methanogens are strictly anaerobic Archaea that derive their energy for growth by reducing a limited number of substrates to methane. "Methanosarcina" spp. utilize the methylotrophic pathway to grow on methanol, methylamines and methylsulfides. These compounds enter the methylotrophic pathway as methyl-coenzyme M, the synthesis of which is mediated by the cooperative action of at least three substrate-specific proteins. "Methanosarcina" spp. carry three copies of the "mtaCB" operon that encode methanol-specific corrinoid protein and methyltransferase 1 (MT1; "mtaCB1," "mtaCB2," and "mtaCB3"), and two copies of "mtaA" that encode methyltransferase 2 (MT2; "mtaA1" and "mtaA2"). Genetic and physiological studies in "M. acetivorans" C2A previously showed the necessity of "mtaA1," dispensability of "mtaA2," and discrete function and differential regulation of "mtaCB" operons on methanol. In this study, we attempted to determine if the "mta" genes encode proteins with different enzymatic properties by constructing various synthetic "mtaCBA" operons that shared conserved transcriptional and translational sequences. When controlled by the strong, tetracycline-regulated P"mcrB"("tetO1"), each copy of "mtaC," "mtaB," and "mtaA" encode fully functional methanol-dependent MT1/MT2 system that exhibited similar growth characteristics on methanol plus tetracycline. Previously reported growth defects on "mtaC3" and "mtaA2" can be mainly attributed to poor expression of these genes on methanol. However, the various synthetic constructs produced unequal amounts of protein that impede quantitative comparison of the enzymatic activities but implicate post-transcriptional regulation. MtaC, MtaB, and MtaA were more abundant in cells grown on methanol where these proteins are required than on trimethylamine where these proteins are not essential. Therefore, the post-transcriptional regulation is physiologically relevant. Each of the "mta" transcripts was expressed at levels ca. 10-fold higher in cells grown on methanol than on TMA, indicating post-transcriptional regulation at the mRNA level. Further studies include identification of the "cis-" and trans-acting elements that contribute to this regulation. Biochemical studies in "M. barkeri" MS previously demonstrated that the synthesis of methyl-CoM also requires an activation protein that maintains the corrinoid protein in its highly reduced Co(I) active state. In "M. barkeri" MS, the "m"ethyltransferase "a"ctivation "p"rotein (MAP) and the "r"eductive "a"ctivation of "m"ethyltransfer, "a"mines (RamA) were previously shown to mediate ATP-dependent reductive reactivation of the methanol- and methylamine-CoM methyltransfer reactions, respectively. However, it remains unclear whether MAP and RamA are distinct, substrate-specific proteins or the same protein that exhibits broad substrate-specificity. In this work, we identified four homologs of "ramA" in "M. acetivorans" C2A that potentially encode substrate-specific corrinoid activation proteins. Genetic analysis indicates that MA0150 ("ramA") encodes an activation protein that is essential for growth on monomethylamine and can recognize multiple corrinoid proteins for the utilization of methanol, trimethylamine, and dimethylamine. MA4380 and MA3972 encode isozymes that can support growth on methanol and DMA. "In silico" and gene expression analyses strongly suggest that MA4380 encodes MAP while MA3972 may play an important role in acetate utilization. MA0849 alone is not sufficient to support growth on methanol, DMA and MMA. The results presented in this study have clearly shown the usefulness of the genetic tractability of "M. acetivorans" in complementing our current understanding of the methylotrophic pathway and in offering more clues to yet undiscovered growth strategies by these metabolically restricted organisms. [The dissertation citations contained here are published with the permission of ProQuest LLC. Further reproduction is prohibited without permission. Copies of dissertations may be obtained by Telephone (800) 1-800-521-0600. Web page:]
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Publication Type: Dissertations/Theses - Doctoral Dissertations
Education Level: N/A
Audience: N/A
Language: English
Sponsor: N/A
Authoring Institution: N/A