SummaryCellular life emerged ~3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles due to the Earth’s rotation. The advantage conferred upon organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation-reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterising their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription-translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular time-keeping with redox homeostatic mechanisms following the Great Oxidation Event ~2.5 billion years ago.

We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodiumproton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.

The environment significantly influences the dynamic expression and assembly of all components encoded in the genome of an organism into functional biological networks. We have constructed a model for this process in Halobacterium salinarum NRC-1 through the data-driven discovery of regulatory and functional interrelationships among approximately 80% of its genes and key abiotic factors in its hypersaline environment. Using relative changes in 72 transcription factors and 9 environmental factors (EFs) this model accurately predicts dynamic transcriptional responses of all these genes in 147 newly collected experiments representing completely novel genetic backgrounds and environments-suggesting a remarkable degree of network completeness. Using this model we have constructed and tested hypotheses critical to this organism's interaction with its changing hypersaline environment. This study supports the claim that the high degree of connectivity within biological and EF networks will enable the construction of similar models for any organism from relatively modest numbers of experiments.

We report the complete sequence of the 4,274,642-bp genome of Haloarcula marismortui, a halophilic archaeal isolate from the Dead Sea. The genome is organized into nine circular replicons of varying G+C compositions ranging from 54% to 62%. Comparison of the genome architectures of Halobacterium sp. NRC-1 and H. marismortui suggests a common ancestor for the two organisms and a genome of significantly reduced size in the former. Both of these halophilic archaea use the same strategy of high surface negative charge of folded proteins as means to circumvent the salting-out phenomenon in a hypersaline cytoplasm. A multitiered annotation approach, including primary sequence similarities, protein family signatures, structure prediction, and a protein function association network, has assigned putative functions for at least 58% of the 4242 predicted proteins, a far larger number than is usually achieved in most newly sequenced microorganisms. Among these assigned functions were genes encoding six opsins, 19 MCP and/or HAMP domain signal transducers, and an unusually large number of environmental response regulators-nearly five times as many as those encoded in Halobacterium sp. NRC-1-suggesting H. marismortui is significantly more physiologically capable of exploiting diverse environments. In comparing the physiologies of the two halophilic archaea, in addition to the expected extensive similarity, we discovered several differences in their metabolic strategies and physiological responses such as distinct pathways for arginine breakdown in each halophile. Finally, as expected from the larger genome, H. marismortui encodes many more functions and seems to have fewer nutritional requirements for survival than does Halobacterium sp. NRC-1.

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ∼64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.

Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals, we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sublethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II), and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large-scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up-regulation of active efflux of Co(II), Ni(II), Cu(II), and Zn(II), down-regulation of Mn(II) uptake and up-regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all of these discoveries into a unified systems-level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals, we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms.[Supplemental material is available online at www.genome.org. The microarray data from this study have been submitted to GEO under accession nos. GSM109343-GSM109461 and GSM109514-GSM109522.]Transition metals such as manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni) copper (Cu), and zinc (Zn) are essential cofactors in the physiology of all organisms. In fact, recent estimates suggest that over half of all proteins in every organism are metalloproteins (Degtyarenko 2000). Although essential in trace amounts, at higher levels these metals can be toxic to cells because they directly or indirectly compromise DNA, protein, and membrane integrity and function. For example, cycling in redox states of metals such as Fe and Cu and antioxidant scavenging by redoxinactive metals such as Zn can both cause oxidative damage to cell components through increased production of reactive oxygen species (ROS) (Nelson 1999). Organisms usually avoid metal toxicity through selective uptake, trafficking, and efflux of metal ions, enzymatic conversion of metals into non-or less-toxic redox states, or sequestering toxic metal ions with ferritins and metallothioneins (Silver 1992;Blindauer et al. 2002;Reindel et al. 2002;Zeth et al. 2004). These mechanisms are believed to be often regulated by free metal-ion concentration (Raab and Feldman 2003). In this regard, other factors such as salinity,...

Cells responding to dramatic environmental changes or undergoing a developmental switch typically change the expression of numerous genes. In bacteria, factors regulate much of this process, whereas in eukaryotes, four RNA polymerases and a multiplicity of generalized transcription factors (GTFs) are required. Here, by using a systems approach, we provide experimental evidence (including protein-coimmunoprecipitation, ChIP-Chip, GTF perturbation and knockout, and measurement of transcriptional changes in these genetically perturbed strains) for how archaea likely accomplish similar large-scale transcriptional segregation and modulation of physiological functions. We are able to associate GTFs to nearly half of all putative promoters and show evidence for at least 7 of the possible 42 functional GTF pairs. This report represents a significant contribution toward closing the gap in our understanding of gene regulation by GTFs for all three domains of life and provides an example for how to use various experimental techniques to rapidly learn significant portions of a global gene regulatory network of organisms for which little has been previously known.archaea ͉ regulatory networks ͉ systems biology T ranscriptional regulation by general transcription factors (GTFs) is an important control point for large-scale regulation of physiology across all three domains of life. It is generally understood that in bacteria, factors help to modulate large changes in gene expression in response to environmental stimuli. Meanwhile, in eukaryotes, families of large multisubunit general transcription complexes initiate large-scale changes in transcription from various promoters. Relatively little is known, however, about how GTFs in archaea help confer fitness across a broad range of environments, including hostile ones, such as thermal vents and hypersaline ponds, to milder environments, such as the oceans and the human oral cavity and gut (1-4).In archaea, two GTFs orthologous to eukaryotic transcription factor IIB (TFB) and TATA-binding proteins (TBPs) are necessary and sufficient for initiating basal transcription (5). These GTFs are present in multiple copies in several archaeal species [supporting information (SI) Table 2]. Halobacterium NRC-1 is particularly complex, because its genome encodes six TBP and seven TFB proteins (6). Because we know that a TBP must pair with a TFB to recruit RNA polymerase to the promoter to form an active preinitiation complex the presence of six TBPs and seven TFBs in Halobacterium NRC-1 theoretically encodes 42 possible TFB-TBP pairs, which may drive transcription from an equally diverse set of promoters in Halobacterium NRC-1Here, we investigate the following: (i) the degree to which members of the two families interact with one another; (ii) whether the promoter specificities of the individual TFBs and TBPs have diverged significantly from one another, or whether they have retained considerable overlap in their genomic binding sites; (iii) whether a higher-order regulatory network architec...

We report a remarkably high UV-radiation resistance in the extremely halophilic archaeon Halobacterium NRC-1 withstanding up to 110 J/m 2 with no loss of viability. Gene knockout analysis in two putative photolyase-like genes (phr1 and phr2) implicated only phr2 in photoreactivation. The UV-response was further characterized by analyzing simultaneously, along with gene function and protein interactions inferred through comparative genomics approaches, mRNA changes for all 2400 genes during light and dark repair. In addition to photoreactivation, three other putative repair mechanisms were identified including d(CTAG) methylation-directed mismatch repair, four oxidative damage repair enzymes, and two proteases for eliminating damaged proteins. Moreover, a UV-induced down-regulation of many important metabolic functions was observed during light repair and seems to be a phenomenon shared by all three domains of life. The systems analysis has facilitated the assignment of putative functions to 26 of 33 key proteins in the UV response through sequence-based methods and/or similarities of their predicted three-dimensional structures to known structures in the PDB. Finally, the systems analysis has raised, through the integration of experimentally determined and computationally inferred data, many experimentally testable hypotheses that describe the metabolic and regulatory networks of Halobacterium NRC-1.[Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to GEO under accession no. GSE1040.]Biological systems have evolved mechanisms to appropriately respond to environmental stresses that can damage proteins and DNA. Halobacterium NRC-1, for example, has evolved the capacity to withstand up to 4.5M salinity, ultraviolet radiation (UV), and oxidative stress in its natural environment. One such adaptation, the high-density of acidic residues on the surface of almost all halobacterial proteins, is believed to stabilize protein structure and function in high salt (Kennedy et al. 2001). It can also physically relocate to favorable environments using sensors for light, oxygen, and nutrients, and produce energy through respiration, fermentation, or phototrophy (Ng et al. 2000). We have previously reported a response mechanism in Halobacterium NRC-1 that coordinately regulates phototrophy and arginine fermentation, two major sources for anaerobic energy production. The effects of perturbing the function of a regulator in this response mechanism were observed throughout the metabolic network (Baliga et al. 2002).Systems approaches allow us to raise a whole series of hypotheses regarding the behavior of a system in response to a defined experimental perturbation. These hypotheses then stimulate iterative cycles of perturbations and analyses to verify these hypotheses. Here, we report a systems-level study on the behavior of Halobacterium NRC-1 upon UV-C irradiation. Shortwave ultraviolet light (UV-C) induces two types of mutagenic lesions in DNA-cyclobutane pyrimid...