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ABSTRACT Gene regulatory networks (GRNs) are critical for dynamic transcriptional responses to environmental stress. However, the mechanisms by which GRN regulation adjusts physiology to enable stress survival remain unclear. Here we investigate the functions of transcription factors (TFs) within the global GRN of the stress-tolerant archaeal microorganism Halobacterium salinarum. We measured growth phenotypes of a panel of TF deletion mutants in high temporal resolution under heat shock, oxidative stress, and low-salinity conditions. To quantitate the noncanonical functional forms of the growth trajectories observed for these mutants, we developed a novel modeling framework based on Gaussian process regression and functional analysis of variance (FANOVA). We employ unique statistical tests to determine the significance of differential growth relative to the growth of the control strain. This analysis recapitulated known TF functions, revealed novel functions, and identified surprising secondary functions for characterized TFs. Strikingly, we observed that the majority of the TFs studied were required for growth under multiple stress conditions, pinpointing regulatory connections between the conditions tested. Correlations between quantitative phenotype trajectories of mutants are predictive of TF-TF connections within the GRN. These phenotypes are strongly concordant with predictions from statistical GRN models inferred from gene expression data alone. With genome-wide and targeted data sets, we provide detailed functional validation of novel TFs required for extreme oxidative stress and heat shock survival. Together, results presented in this study suggest that many TFs function under multiple conditions, thereby revealing high interconnectivity within the GRN and identifying the specific TFs required for communication between networks responding to disparate stressors. IMPORTANCE To ensure survival in the face of stress, microorganisms employ inducible damage repair pathways regulated by extensive and complex gene networks. Many archaea, microorganisms of the third domain of life, persist under extremes of temperature, salinity, and pH and under other conditions. In order to understand the cause-effect relationships between the dynamic function of the stress network and ultimate physiological consequences, this study characterized the physiological role of nearly one-third of all regulatory proteins known as transcription factors (TFs) in an archaeal organism. Using a unique quantitative phenotyping approach, we discovered functions for many novel TFs and revealed important secondary functions for known TFs. Surprisingly, many TFs are required for resisting multiple stressors, suggesting cross-regulation of stress responses. Through extensive validation experiments, we map the physiological roles of these novel TFs in stress response back to their position in the regulatory network wiring. This study advances understanding of the mechanisms underlying how microorganisms resist extreme stress. Given the generality of the methods employed, we expect that this study will enable future studies on how regulatory networks adjust cellular physiology in a diversity of organisms.

Networks of interacting transcription factors are central to the regulation of cellular responses to abiotic stress. Although the architecture of many such networks has been mapped, their dynamic function remains unclear. Here we address this challenge in archaea, microorganisms possessing transcription factors that resemble those of both eukaryotes and bacteria. Using genome-wide DNA binding location analysis integrated with gene expression and cell physiological data, we demonstrate that a bacterial-type transcription factor (TF), called RosR, and five TFIIB proteins, homologs of eukaryotic TFs, combinatorially regulate over 100 target genes important for the response to extremely high levels of peroxide. These genes include 20 other transcription factors and oxidative damage repair genes. RosR promoter occupancy is surprisingly dynamic, with the pattern of target gene expression during the transition from rapid growth to stress correlating strongly with the pattern of dynamic binding. We conclude that a hierarchical regulatory network orchestrated by TFs of hybrid lineage enables dynamic response and survival under extreme stress in archaea. This raises questions regarding the evolutionary trajectory of gene networks in response to stress.