IMPACT OF THERMAL AND CHEMICAL STRESS ON SULFOLOBUS SOLFATARICUS P2 GENOME AND COMPONENTS OF TRANSCRIPTION APPARATUS

Abstract

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, and TBP- interacting protein-49 (TIP49) 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. In this study it was hypothesised that stress modulates the expression and/or stability of one or more components of Sulfolobus transcriptional apparatus. To test this, the cellular and biochemical consequences of subjecting Sulfolobus solfataricus P2 to chemical and thermal stresses as well as their effects on Sulfolobus transcription machinery were studied. Results show that elevating the temperature from 76 ̊C to iv90 ̊C (heat shock) for 5 minutes results in large scale protein aggregation and altered cellular morphology of Sulfolobus heat shocked cells. Moreover, immunochemical analyses suggested that TFE levels in heat shocked cells experience a rapid decline while its mRNA levels continue to rise even after 30 minutes of heat shock. Furthermore, 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 M hydrogen peroxide is detrimental. Specifically, cells treated with 2% isopropanol or 200M hydrogen peroxide alter their morphologies and harbour degraded genomes as well as proteomes that are partially depleted. Isopropanol and hydrogen peroxide exposure does not promote random protein degradation but instead preferentially impacts fates of certain transcription factors. Whereas isopropanol mediated degradation of genomic DNA in Sulfolobus cells is not affected by EDTA, oxidative stress-induced genomic breakdown is inhibited with EDTA. Moreover, the damaging effects of 2% isopropanol or 200 M H 2 O 2 on host genome and proteome are restricted to Sulfolobus and are not observed in either bacterial or cultured eukaryotic human cells. Taken together, these results demonstrate that in Sulfolobus solfataricus P2 cells: 1) TFE is depleted by heat shock and does not appear to function as a general transcription factor, 2) thermal and chemical stresses impact the stability of TBP, TFB1, TFE, TIP49 and RpoB differentially, and 3) isopropanol and hydrogen peroxide mediated genomic DNA degradation is observed only in archaeal cells and likely occurs through different mechanisms.