University of Illinois Urbana-Champaign

Insights into substrate utilization and transcriptional regulation in methanosarcina

Lopez Munoz, Madeline Mayrim

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LOPEZMUNOZ-DISSERTATION-2017.pdf

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https://hdl.handle.net/2142/97657

Description

Title
Insights into substrate utilization and transcriptional regulation in methanosarcina
Author(s)
Lopez Munoz, Madeline Mayrim
Issue Date
2017-02-20
Director of Research (if dissertation) or Advisor (if thesis)
Metcalf, William M
Doctoral Committee Chair(s)
Metcalf, William M
Committee Member(s)
  • Whitaker, Rachel J.
  • Farrand, Stephen K.
  • Cann, Isaac
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2017-08-10T20:32:32Z
Keyword(s)
  • Methanogens
  • Archaea
  • Metabolism
  • Pyruvate
  • Transcription regulation
Abstract
Methanogenic archaea are strictly anaerobic organisms responsible for the production of all biologically produced methane making them key players of the global carbon cycle. These unusual organisms derive their energy from the reduction of one-carbon (C-1) compounds to methane. Among methanogenic archaea, Methanosarcina are the most metabolically diverse being able to use H2/CO2, acetate, methanol, methylamines and methyl sulfides as substrates for methanogenesis. This dissertation presents biochemical and genetic studies on pyruvate utilization in Methanosarcina as a substrate for methanogenesis and the regulation of methylotrophic methanogenesis in M. acetivorans in an effort to further delineate the intrinsic metabolic diversity found in Methanosarcina species. Organic compounds such as fatty acids and sugars are rarely substrates for methanogenesis unless they are co-metabolized by syntrophic associations. Previously, an M. barkeri mutant that had the capacity to use pyruvate as substrate was isolated. Chapter 2 of this dissertation is focused towards understanding the molecular basis of pyruvate utilization in these cells. Genetic and transcriptomic studies on two cell lines of the mutant strain revealed the presence of two mutations in both strains. These changes in genome sequence resulted in cells that over-expressed pyruvate ferredoxin oxidoreductase (Por) an enzyme responsible for the conversion of pyruvate to acetyl-CoA and lacked pyruvate carboxylase activity, the enzyme responsible for synthesis of oxaloacetate in the cells. Biochemical and transcriptomic experiments revealed that in order to make the central metabolite oxaloacetate this mutant was using an alternative reaction catalyzed by phosphoenolpyruvate carboxylase. Surprisingly, our results revealed that Por is essential in M. bakeri suggesting this enzyme to have an alternative function to pyruvate synthesis in the cells. Chapter three entails the characterization of the regulatory network that controls methylotrophic methanogenesis, the pathway used for growth on methylated compounds like methanol, methylamines and methyl sulfides. Methylated compounds enter the methanogenesis pathway at the level of methyl-coenzyme M, the synthesis of which is mediated by the substrate specific methyltransferase complex MT1 and MT2. Growth on methanol requires the MT1 complex encoded by the mtaCB1 operon. Previous work showed that this MT1 complex is among the highest differentially regulated genes in Archaea and that its transcription is regulated by the methanol specific regulators msrA and msrB. Biochemical and transcriptomic data presented in this dissertation revealed that MsrA and MsrB are DNA binding proteins that activate the transcription of mtaCB1 by binding to its promoter as heterodimers. Transcriptomic data also revealed that MsrB could act as both repressor and activator of transcription, since its deletion caused an increase in expression of methylamine methyltransferase genes. Complementation of msrB deletion strains resulted in restoration of regulation, thus confirming the role of msrB in repression of methylamine methyltransferase genes. Finally, Chapter 4 of this dissertation is focused towards understating how Methanosarcina cells are able to sense and respond to environmental signals in order to activate the substrate specific methyltransferase genes. Bioinformatic analysis revealed the presence of three putative histidine kinases (MA0863, MA4377, MA4377), a response regulator (MA4376) and an msr-like protein (MA4375) that due to their genomic localization could be involved in the regulation of methyl sulfide methyltransferase (mts) genes. Deletion and transcriptomic studies revealed that the three-histidine kinases may be involved in the regulation of three methyl sulfide methyltransferases and that the single response regulator MA4376 may to be interacting with more than one of these histidine kinases in order to regulate mts genes.
Graduation Semester
2017-05
Type of Resource
text
Permalink
http://hdl.handle.net/2142/97657
Copyright and License Information
Copyright 2017 Madeline M. López Muñoz

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