Functional Assessment of Transcriptional Regulation in the Methanogen, Methanosarcina acetivorans

Open Access
Author:
Reichlen, Matthew James
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
March 24, 2010
Committee Members:
  • James Gregory Ferry, Dissertation Advisor
  • James Gregory Ferry, Committee Chair
  • Katsuhiko Murakami, Committee Chair
  • Sarah Ellen Ades, Committee Member
  • Benjamin Franklin Pugh, Committee Member
  • Philip C. Bevilacqua, Committee Member
Keywords:
  • Archaea
  • Transcription
  • Methanogen
  • Methanosarcina
  • acetivorans
Abstract:
The methanogenic archaea play an essential role in the biosphere by controlling the flux of carbon between oxic and anoxic environments. Yet much of the basic the physiology of these organisms is poorly understood. This dissertation strives to further the understanding of gene regulation in the methanogenic archaea using the model organism, Methanosarcina acetivorans. The first two chapters provide overviews of the methanogens and of the current understanding on archaeal transcription. The final four chapters describe the experimental work performed. A functional investigation of the three TATA-binding protein (TBP) homologs in M. acetivorans is described in chapter 3. TBP1 was found to function as the primary general transcription factor (GTF) and was essential for M. acetivorans growth. The minor TBPs, TBP2 and TBP3 were dispensable for growth but required for optimal fitness during culturing with acetate likely due to a role in the optimization of gene expression. Chapter 4 analyzes the transcriptional response of M. acetivorans diauxic growth with methanol and acetate. This analysis of a metabolic transition, the first of its kind for a methanogen, significantly contributed to the understanding of M. acetivorans physiology and ascribed potential biological roles to numerous previously uncharacterized or poorly characterized genes. The fifth chapter describes a functional investigation of MA3302, a member of a widespread yet uncharacterized family of euryarchaeal transcription factors. MA3302 expression was elevated during acetoclastic growth, was required for optimal growth on this substrate and was highly conserved in acetotrophic methanogens consistent with an involvement in transcriptional regulation during growth on acetate. Future investigations will seek to identify target genes for regulation by MA3302 and to overexpress, purify and characterize the recombinant MA3302 protein. Ongoing investigations addressing the roles of the three M. acetivorans TBP homologs are presented in the final chapter. Unresolved issues under investigation include assessments of why tbp1 is essential for M. acetivorans growth and of physiological consequence resulting from the simultaneous deletion of tbp2 and tbp3 and to identify DNA-binding loci for each TBP isomer on the M. acetivorans chromosome.