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|Title:||Analysis of the MT1/MT2 Systems Involved in the Metabolism of One-Carbon Compounds in Methanosarcina Acetivorans C2A|
|Author(s):||Opulencia, Rina Bagsic|
|Doctoral Committee Chair(s):||Metcalf, William W.|
|Department / Program:||Microbiology|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||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 PmcrB( 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&barbelow;ethyltransferase a&barbelow;ctivation p&barbelow;rotein (MAP) and the r&barbelow;eductive a&barbelow;ctivation of m&barbelow;ethyltransfer, a&barbelow;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.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.
|Date Available in IDEALS:||2014-12-17|