Functional Genomics of Stress Response in the Hyperthermophilic Crenarchaeon Sufolobus solfataricus and Role of vapBC Toxin-Antitoxin Loci in RNA Management.

TACHDJIAN, SABRINA. Functional Genomics of Stress Response in the Hyperthermophilic Crenarchaeon Sufolobus solfataricus and Role of vapBC ToxinAntitoxin Loci in RNA Management. (Under the direction of Dr. R.M. Kelly.) Functional genomics tools were used to examine the dynamic stress response of the hyperthermophile Sulfolobus solfataricus subjected to thermal and pH shifts relative to growth under normal conditions. Whole genome oligonucleotide microarray analyses revealed substantial transcriptional changes in this crenarcheon upon temperature shifts from 80 to 90°C at pH 4.0, and pH shifts from 4.0 to 2.0 at 80°C. ORFs encoding heat shock proteins, molecular chaperones, and other known stress response elements responded in expected ways. However, genes involved in membrane adaptation, DNA repair mechanisms, transcriptional regulation, were also impacted, indicating that stress response is complex and implicating metabolic adjustments that go beyond the protective effect of stress proteins. It was also observed that, especially under heat shock, genes encoding insertion sequence (IS) elements were massively induced, suggesting that this microorganism increases genome plasticity as a strategy for dealing with thermal stress. In addition, 10 ORFs belonging to vapBC Toxin-Antitoxin (TA) loci were also implicated in the stress response of this archaeon. In silico analysis of the structural features of S. solfataricus two VapC toxins was conducted to examine the putative roles of Toxin-Antitoxin (TA) systems with respect to RNA management. Superimposition of predicted VapC structures on the T4 RNAse H template showed conservation of key catalytic residues as well as active site conformation, supporting their designation as ribonucleases. To confirm this biochemically, a recombinant version of VapBC-22, a heat shock-inducible and highly transcribed TA pair in S. solfataricus, was produced in Escherichia coli. Mass spectrometry confirmed that both proteins encoded by this locus could be recovered in soluble forms and that they associate in vitro. Moreover, recombinant VapC-22 exhibited ribonucleic activity. A vapC-22 disruption mutant was constructed and transcriptional response analysis showed that toxin impacted RNA metabolism and translational functions under thermal stress. Possible connections to a putative archaeal RNA interference and/or non-sense mediated decay pathway were proposed. Furthermore, the connection between VapBC TA systems and translation inhibition was strengthened by the in silico identification of a pattern conserved in most archaeal genome sequences, whereby the gene encoding the gamma subunit of translation initiation factor 2 was found to overlap with a vapBC locus. The results of this study implicate vapBC loci in RNA management and suggest the intriguing possibility that TA systems are connected to RNAi/NMD pathways in S. solfataricus and other archaea. Functional Genomics of Stress Response in the Hyperthermophilic Crenarchaeon Sufolobus solfataricus and Role of vapBC Toxin-Antitoxin Loci in RNA Management By SABRINA TACHDJIAN A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy. Chemical and Biomolecular Engineering Raleigh, NC July, 2007