Inference of an environmental and gene regulatory influence network (EGRINOS) by integrating transcriptional responses to H2O2 and paraquat (PQ) has revealed a multi-tiered oxidative stress (OS)-management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, production of rhodopsins, carotenoids and gas vesicles, metal trafficking, and various other aspects of metabolism.ChIP-chip, microarray, and survival assays have validated important architectural aspects of this network, identified novel defense mechanisms (including two evolutionarily distant peroxidase enxymes), and showed that general transcription factors of the transcription factor B family have an important function in coordinating the OS response (OSR) despite their inability to directly sense ROS.A comparison of transcriptional responses to sub-lethal doses of H2O2 and PQ with predictions of these responses made by an EGRIN model generated earlier from responses to other environmental factors has confirmed that a significant fraction of the OSR is made up of a generalized component that is also observed in response to other stressors.Analysis of active regulons within environment and gene regulatory influence network for OS (EGRINOS) across diverse environmental conditions has identified the specialized component of oxidative stress response (OSR) that is triggered by sub-lethal OS, but not by other stressors, including sub-inhibitory levels of redox-active metals, extreme changes in oxygen tension, and a sub-lethal dose of γ rays.
Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide (O2−), and hydroxyl (OH−) radicals, are normal by-products of aerobic metabolism. Evolutionarily conserved mechanisms including detoxification enzymes (peroxidase/catalase and superoxide dismutase (SOD)) and free radical scavengers manage this endogenous production of ROS. OS is a condition reached when certain environmental stresses or genetic defects cause the production of ROS to exceed the management capacity. The damage to diverse cellular components including DNA, proteins, lipids, and carbohydrates resulting from OS (Imlay, 2003; Apel and Hirt, 2004; Perrone et al, 2008) is recognized as an important player in many diseases and in the aging process (Finkel, 2005).
We have applied a systems approach to characterize the OSR of an archaeal model organism, Halobacterium salinarum NRC-1. This haloarchaeon grows aerobically at 4.3 M salt concentration in which it routinely faces cycles of desiccation and rehydration, and increased ultraviolet radiation—both of which can increase the production of ROS (Farr and Kogoma, 1991; Oliver et al, 2001). We have reconstructed the physiological adjustments associated with management of excessive OS through the analysis of global transcriptional changes elicited by step exposure to growth sub-inhibitory and sub-lethal levels of H2O2 and PQ (a redox-cycling drug that produces O2−; Hassan and Fridovich, 1979) as well as during subsequent recovery from these stresses. We have integrated all of these data into a unified model for OSR to discover conditional functional links between protective mechanisms and normal aspects of metabolism. Subsequent phenotypic analysis of gene deletion strains has verified the conditional detoxification functions of three putative peroxidase/catalase enzymes, two SODs, and the protective function of rhodopsins under increased levels of H2O2 and PQ. Similarly, we have also validated ROS scavenging by carotenoids and flotation by gas vesicles as secondary mechanisms that may minimize OS.
Given the ubiquitous nature of OS, it is not entirely surprising that most organisms have evolved similar multiple lines of defense—both passive and active. Although such mechanisms have been extensively characterized using other model organisms, our integrated systems approach has uncovered additional protective mechanisms in H. salinarum (e.g. two evolutionarily distant peroxidase/catalase enzymes) and revealed a structure and hierarchy to the OSR through conditional regulatory associations among various components of the response. We have validated some aspects of the architecture of the regulatory network for managing OS by confirming physical protein–DNA interactions of six transcription factors (TFs) with promoters of genes they were predicted to influence in EGRINOS. Furthermore, we have also shown the consequence of deleting two of these TFs on transcript levels of genes they control and survival rate under OS. It is notable that these TFs are not directly associated with sensing ROS, but, rather, they have a general function in coordinating the overall response. This insight would not have been possible without constructing EGRINOS through systems integration of diverse datasets.
Although it has been known that OS is a component of diverse environmental stress conditions, we quantitatively show for the first time that much of the transcriptional responses induced by the two treatments could indeed have been predicted using a model constructed from the analysis of transcriptional responses to changes in other environmental factors (UV and γ-radiation, light, oxygen, and six metals). However, using specific examples we also reveal the specific components of the OSR that are triggered only under severe OS. Notably, this model of OSR gives a unified perspective of the interconnections among all of these generalized and OS-specific regulatory mechanisms.
Complexity of cellular response to oxidative stress (OS) stems from its wide-ranging damage to nucleic acids, proteins, carbohydrates, and lipids. We have constructed a systems model of OS response (OSR) for Halobacterium salinarum NRC-1 in an attempt to understand the architecture of its regulatory network that coordinates this complex response. This has revealed a multi-tiered OS-management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, production of rhodopsins, carotenoids and gas vesicles, metal trafficking, and various other aspects of metabolism. Through experimental validation of interactions within the OSR regulatory network, we show that despite their inability to directly sense reactive oxygen species, general transcription factors have an important function in coordinating this response. Remarkably, a significant fraction of this OSR was accurately recapitulated by a model that was earlier constructed from cellular responses to diverse environmental perturbations—this constitutes the general stress response component. Notwithstanding this observation, comparison of the two models has identified the coordination of frontline defense and repair systems by regulatory mechanisms that are triggered uniquely by severe OS and not by other environmental stressors, including sub-inhibitory levels of redox-active metals, extreme changes in oxygen tension, and a sub-lethal dose of γ rays.