COMPARATIVE ANALYSIS OF TRANSCRIPTIONAL RESPONSE TO STRESS AND CARBOHYDRATE AVAILABILITY IN HALOARCHAEA
dc.contributor.advisor | Schmid, Amy K | |
dc.contributor.advisor | Wray, Gregory A | |
dc.contributor.author | Hackley, Rylee K | |
dc.date.accessioned | 2024-03-07T18:39:25Z | |
dc.date.available | 2024-03-07T18:39:25Z | |
dc.date.issued | 2023 | |
dc.department | Genetics and Genomics | |
dc.description.abstract | Hypersaline-adapted archaea, or haloarchaea, inhabit extreme environments where changes in near-saturated salinity, oxygen, and nutrients require rapid regulation of essential cellular processes. Transcriptional regulatory networks govern the dynamical responses enabling these organisms to sense and respond to rapidly changing conditions. Additionally, timely regulation of carbon metabolic pathways is essential to respond to intermittent nutrient availability and prevent futile cycling of intracellular metabolites. In Halobacterium salinarum, a hypersaline-adapted archaeon that does not rely on carbohydrates for carbon or energy, over 1,500 transcriptome profiling experiments have yielded a genome-scale model of regulatory interactions and revealed a general transcriptional response to diverse stressors (Chapter 2). Among regulators in Hbt. salinarum, a sugar-sensing TrmB family protein has previously been shown to control gluconeogenesis and other biosynthetic pathways. This characterization expanded the set of regulatory functions known for TrmB-family proteins in archaea, which regulate carbohydrate metabolism in hyperthermophilic archaea. TrmB regulators are particularly interesting in Haloarchaea because an expansion of the protein family is presumed to have occurred alongside a diversification of carbohydrate catabolic pathways (Chapter 3). To investigate whether the expanded set of TrmB functions is shared among haloarchaea with different metabolic capabilities and better understand how regulatory variation arises in extremophiles, we characterized the role of TrmB in two haloarchaeal model species that catabolize carbohydrates: Haloarcula hispanica and Haloferax volcanii (Chapters 4 and 5, respectively). We hypothesized that TrmB would maintain a role in the regulation of gluconeogenesis through homologous targets but would acquire targets involved in the concordant catabolic processes of these saccharolytic models. To characterize the role of TrmB homologs, we conducted high-throughput growth assays, microscopy and other microbiological phenotyping techniques, gene expression profiling via RNA-seq, promoter activity analysis, and protein-DNA binding assays of TrmB homologs in Har. hispanica and Hfx. volcanii. Our results show that TrmB homologs indeed activate gluconeogenesis through the recognition of conserved cis-regulatory motifs. However, contrary to its role in Hbt. salinarum, TrmB does not act as a global regulator in Har. hispanica or Hfx. volcanii: it does not directly repress the expression of peripheral pathways such as cofactor biosynthesis or catabolism of other carbon sources. A key bidirectional control point, activation of ppsA and repression of pyk, is lost in Har. hispanica. Our results indicate substantial rewiring of the TrmB regulon in Hfx. volcanii. A novel transcriptional regulator, TbsP, is responsible for repressing gluconeogenic genes when glucose is available. TrmB and TbsP appear to compete for partially overlapping binding sites in the promoter of gapII, which encodes the gluconeogenic-specific glyceraldehyde-3-phosphate dehydrogenase. Loss-of-function mutations in tbsP are sufficient to recover partial gapII expression and gluconeogenic activity when trmB is deleted. In Hfx. volcanii, TrmB is predicted to activate ppsA and repress the gene encoding bacterial phosphoenopyruvate carboxylase, perhaps preserving the dynamical and functional behavior of TrmB in Hbt salinarum, but reflecting species-specific anaplerotic strategies. Moreover, TrmB is predicted to repress the expression of bacterial type I GAPDH: gapI and gapII may comprise an additional bidirectional control point in Hfx. volcanii, although this hypothesis requires additional testing. Cumulatively, this dissertation outlines specific examples of TRN rewiring highlighting the incorporation of metabolic enzymes gained through inter-domain horizontal gene transfer, suggesting rewiring of the TrmB regulon via gain and loss of binding sites alongside metabolic network evolution in Haloarchaea. | |
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dc.rights.uri | ||
dc.subject | Microbiology | |
dc.subject | Genetics | |
dc.subject | Molecular biology | |
dc.subject | Archaea | |
dc.subject | gluconeogensis | |
dc.subject | Haloarchaea | |
dc.subject | microbial metabolism | |
dc.subject | Transcriptional regulation | |
dc.subject | transcriptional regulatory networks | |
dc.title | COMPARATIVE ANALYSIS OF TRANSCRIPTIONAL RESPONSE TO STRESS AND CARBOHYDRATE AVAILABILITY IN HALOARCHAEA | |
dc.type | Dissertation |
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