Impact of thermal and chemical stress on sulfolobus solfactaricus P2 genome and components of transcription apparatus
Date of Award
Doctor of Philosophy in Health Science (PhD)
Biological and Biomedical Sciences
Archaea represent the third domain of life and comprise a highly diverse class of microorganisms some of which can withstand extremes of temperature, pressure, pH and salinity. It is for this reason that members of this group are also collectively referred to as "extremophiles". Archaea have a number of unique features such as ether-linked lipids in their cell membranes but also share several important characteristics with eukaryotes and bacteria. For example, like bacteria archaeal genomes are circular but their gene promoters and transcriptional apparatus is more closely related to the eukaryotic RNA polymerase-II system. In Sulfolobus, a model crenarchaeote, transcription is dependent on TATA binding protein (TBP), transcription factor-B1 (TFB1), and perhaps also on transcription factor-E (TFE) which serve as specificity factors for the 12 subunit RNA polymerase. Sulfolobus genome also encodes for other putative transcription factors such as TFB2, TBPinteracting protein-49 (TIP49), and transcription factor-S (TFS) whose functions remain elusive. All cells are capable of coping with transient thermal and chemical stresses by triggering expression of discrete sets of genes whose products prevent cell death. Such responses have been well documented in bacteria and eukaryotes but the effect(s) of such insults on cell morphology, proteome, genome, transcription as well as on the fates of various components of transcription in archaea remain unknown. Here, I have studied the cellular and biochemical consequences of subjecting Sulfolobus solfataricus P2 to chemical and thermal stress. My results show that elevating the temperature from 76°C to 90°C (heat shock) for 5 minutes results in large scale protein aggregation which results in the emergence of large whitish regions in the middle or periphery of heat shocked cells. Moreover, using antibodies generated against recombinant TBP, TFB1, TFE, TIP49 and RpoB (largest subunit of RNA polymerase), I find that TFE levels in heat shocked cells experience a rapid decline while its mRNA levels as well as that of TFB I continued to rise even after 30 minutes of heat shock. Such declines in TFE, or any of the other factors, are not observed in cells that are subjected to DNA damage, mild oxidative stress, or coldshock. Moreover, temperature-shift experiments demonstrate that outgrowth of heat shocked cells is dependent on restoration of TFE levels. While heat shock promotes selective depletion of TFE and does not affect genomic or proteomic integrity to any significant extent, exposure of cells to >0.25% isopropanol or >100 μM hydrogen peroxide is detrimental. Specifically, cells treated with 2% isopropanol or 20014M hydrogen peroxide have altered morphologies and contain degraded genomes and almost depleted proteomes. isopropanol exposure does not influence TBP or TIP49 protein levels but causes depletion of TFB1. TFE, and RpoB at temperatures >76°C. Additionally, subjecting cells to heavy oxidative stress at different temperatures promotes complete genomic DNA breakdown at 50°C and results in rapid decline of TIP49 and. RpoB levels at <76°C. Whereas isopropanol mediated degradation of genomic DNA in Sulfolobus cells is not affected by EDTA, oxidative stress-induced genomic breakdown can he inhibited with by EDTA. Moreover, the damaging effects of 2% isopropanol or 2001.1M I-1202 on host genome and proteome are restricted to Sulfolobus (and perhaps other archaea) and are not observed in either bacterial or eukaryal cells. Taken together, my results demonstrate that in Sulfolobus sollataricus P2 cells: 1) TFE is depleted by heat shock and does not function as a general transcription factor. 2) thermal and chemical stresses impact the stability of TBP, TFB1, TFE, T1P49 and RpoB differentially, and 3) isopropanol and hydrogen peroxide mediated genomic DNA degradation is observed only in only archaeal cells and likely occurs through different mechanisms.
Iqbal, J. (2011). Impact of thermal and chemical stress on sulfolobus solfactaricus P2 genome and components of transcription apparatus (Unpublished doctoral thesis). Aga Khan University, Karachi, Pakistan.