Global Transcriptional Programs in Archaea Share Features with the Eukaryotic Environmental Stress Response.
ABSTRACT: The environmental stress response (ESR), a global transcriptional program originally identified in yeast, is characterized by a rapid and transient transcriptional response composed of large, oppositely regulated gene clusters. Genes induced during the ESR encode core components of stress tolerance, macromolecular repair, and maintenance of homeostasis. In this review, we investigate the possibility for conservation of the ESR across the eukaryotic and archaeal domains of life. We first re-analyze existing transcriptomics data sets to illustrate that a similar transcriptional response is identifiable in Halobacterium salinarum, an archaeal model organism. To substantiate the archaeal ESR, we calculated gene-by-gene correlations, gene function enrichment, and comparison of temporal dynamics. We note reported examples of variation in the ESR across fungi, then synthesize high-level trends present in expression data of other archaeal species. In particular, we emphasize the need for additional high-throughput time series expression data to further characterize stress-responsive transcriptional programs in the Archaea. Together, this review explores an open question regarding features of global transcriptional stress response programs shared across domains of life.
Project description:Previous work has shown that the hypersaline-adapted archaeon, Halobacterium salinarum NRC-1, is highly resistant to oxidative stress caused by exposure to hydrogen peroxide, UV, and gamma radiation. Dynamic alteration of the gene regulatory network (GRN) has been implicated in such resistance. However, the molecular functions of transcription regulatory proteins involved in this response remain unknown.Here we have reanalyzed several existing GRN and systems biology datasets for H. salinarum to identify and characterize a novel winged helix-turn-helix transcription factor, VNG0258H, as a regulator required for reactive oxygen species resistance in this organism. This protein appears to be unique to the haloarchaea at the primary sequence level. High throughput quantitative growth assays in a deletion mutant strain implicate VNG0258H in extreme oxidative stress resistance. According to time course gene expression analyses, this transcription factor is required for the appropriate dynamic response of nearly 300 genes to reactive oxygen species damage from paraquat and hydrogen peroxide. These genes are predicted to function in repair of oxidative damage to proteins and DNA. In vivo DNA binding assays demonstrate that VNG0258H binds DNA to mediate gene regulation.Together these results suggest that VNG0258H is a novel archaeal transcription factor that regulates gene expression to enable adaptation to the extremely oxidative, hypersaline niche of H. salinarum. We have therefore renamed VNG0258H as RosR, for reactive oxygen species regulator.
Project description:A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs) are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq) and a primary-transcript enriched (dRNA-seq) strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (?7% of the annotated genome). Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.
Project description:Widespread microbial genome sequencing presents an opportunity to understand the gene regulatory networks of non-model organisms. This requires knowledge of the binding sites for transcription factors whose DNA-binding properties are unknown or difficult to infer. We adapted a protein structure-based method to predict the specificities and putative regulons of homologous transcription factors across diverse species. As a proof-of-concept we predicted the specificities and transcriptional target genes of divergent archaeal feast/famine regulatory proteins, several of which are encoded in the genome of Halobacterium salinarum. This was validated by comparison to experimentally determined specificities for transcription factors in distantly related extremophiles, chromatin immunoprecipitation experiments, and cis-regulatory sequence conservation across eighteen related species of halobacteria. Through this analysis we were able to infer that Halobacterium salinarum employs a divergent local trans-regulatory strategy to regulate genes (carA and carB) involved in arginine and pyrimidine metabolism, whereas Escherichia coli employs an operon. The prediction of gene regulatory binding sites using structure-based methods is useful for the inference of gene regulatory relationships in new species that are otherwise difficult to infer.
Project description:Halobacterium salinarum are halophilic archaea that display directional swimming in response to various environmental signals, including light, chemicals and oxygen. In Hbt. salinarum, the building blocks (archaellins) of the archaeal swimming apparatus (the archaellum) are N-glycosylated. However, the physiological importance of archaellin N-glycosylation remains unclear. Here, a tetrasaccharide comprising a hexose and three hexuronic acids decorating the five archaellins was characterized by mass spectrometry. Such analysis failed to detect sulfation of the hexuronic acids, in contrast to earlier reports. To better understand the physiological significance of Hbt. salinarum archaellin N-glycosylation, a strain deleted of aglB, encoding the archaeal oligosaccharyltransferase, was generated. In this ?aglB strain, archaella were not detected and only low levels of archaellins were released into the medium, in contrast to what occurs with the parent strain. Mass spectrometry analysis of the archaellins in ?aglB cultures did not detect N-glycosylation. ?aglB cells also showed a slight growth defect and were impaired for motility. Quantitative real-time PCR analysis revealed dramatically reduced transcript levels of archaellin-encoding genes in the mutant strain, suggesting that N-glycosylation is important for archaellin transcription, with downstream effects on archaellum assembly and function. Control of AglB-dependent post-translational modification of archaellins could thus reflect a previously unrecognized route for regulating Hbt. salinarum motility.
Project description:Microbes can tailor transcriptional responses to diverse environmental challenges despite having streamlined genomes and a limited number of regulators. Here, we present data-driven models that capture the dynamic interplay of the environment and genome-encoded regulatory programs of two types of prokaryotes: Escherichia coli (a bacterium) and Halobacterium salinarum (an archaeon). The models reveal how the genome-wide distributions of cis-acting gene regulatory elements and the conditional influences of transcription factors at each of those elements encode programs for eliciting a wide array of environment-specific responses. We demonstrate how these programs partition transcriptional regulation of genes within regulons and operons to re-organize gene-gene functional associations in each environment. The models capture fitness-relevant co-regulation by different transcriptional control mechanisms acting across the entire genome, to define a generalized, system-level organizing principle for prokaryotic gene regulatory networks that goes well beyond existing paradigms of gene regulation. An online resource (http://egrin2.systemsbiology.net) has been developed to facilitate multiscale exploration of conditional gene regulation in the two prokaryotes.
Project description:Multiple general transcription factors (GTFs), TBP and TFB, are present in many haloarchaea, and are deemed to accomplish global gene regulation. However, details and the role of GTF-directed transcriptional regulation in stress response are still not clear. Here, we report a comprehensive investigation of the regulatory mechanism of a heat-induced gene (hsp5) from Halobacterium salinarum. We demonstrated by mutation analysis that the sequences 5' and 3' to the core elements (TATA box and BRE) of the hsp5 promoter (P(hsp5)) did not significantly affect the basal and heat-induced gene expression, as long as the transcription initiation site was not altered. Moreover, the BRE and TATA box of P(hsp5) were sufficient to render a nonheat-responsive promoter heat-inducible, in both Haloferax volcanii and Halobacterium sp. NRC-1. DNA-protein interactions revealed that two heat-inducible GTFs, TFB2 from H. volcanii and TFBb from Halobacterium sp. NRC-1, could specifically bind to P(hsp5) likely in a temperature-dependent manner. Taken together, the heat-responsiveness of P(hsp5) was mainly ascribed to the core promoter elements that were efficiently recognized by specific heat-induced GTFs at elevated temperature, thus providing a new paradigm for GTF-directed gene regulation in the domain of Archaea.
Project description:Genomic analysis points to N-glycosylation as being a common posttranslational modification in Archaea. To date, however, pathways of archaeal N-glycosylation have only been described for few species. With this in mind, the similarities of N-linked glycans decorating glycoproteins in the haloarchaea Haloferax volcanii and Halobacterium salinarum directed a series of bioinformatics, genetic, and biochemical experiments designed to describe that Hbt. salinarum pathway responsible for biogenesis of one of the two N-linked oligosaccharides described in this species. As in Hfx. volcanii, where agl (archaeal glycosylation) genes that encode proteins responsible for the assembly and attachment of a pentasaccharide to target protein Asn residues are clustered in the genome, Hbt. salinarum also contains a group of clustered homologous genes (VNG1048G-VNG1068G). Introduction of these Hbt. salinarum genes into Hfx. volcanii mutant strains deleted of the homologous sequence restored the lost activity. Moreover, transcription of the Hbt. salinarum genes in the native host, as well as in vitro biochemical confirmation of the predicted functions of several of the products of these genes provided further support for assignments made following bioinformatics and genetic experiments. Based on the results obtained in this study, the first description of an N-glycosylation pathway in Hbt. salinarum is offered.
Project description:Halophilic archaea are known to produce a diverse array of pigments for phototrophy and photoprotection. The aim of this paper was to determine the role of a Halobacterium gene encoding the predicted cytochrome P450 monooxygenase (CYP174A1) in pigment synthesis through a combined genetic, phenotypic, and transcriptomic approach. We report on the observed phenotype changes [increased bacterioruberin levels and the loss of purple membrane (PM)] between the Halobacterium salinarum R1 and its CYP174A1-deletion mutant. In addition, we report on the whole-genome DNA microarray analysis, which supports the phenotype of PM loss. This work expands our understanding of the bop-gene regulon, and its relation to carotenoid biosynthesis, and sheds light on our broader understanding of the role (s) of CYP174A1 in archaeal pigment synthesis. To date, this is the first study in which the physiological role of any cytochrome P450 monooxygenase (CYP450) in extremely halophilic archaea has been reported.
Project description:Microbial growth curves are used to study differential effects of media, genetics, and stress on microbial population growth. Consequently, many modeling frameworks exist to capture microbial population growth measurements. However, current models are designed to quantify growth under conditions for which growth has a specific functional form. Extensions to these models are required to quantify the effects of perturbations, which often exhibit nonstandard growth curves. Rather than assume specific functional forms for experimental perturbations, we developed a general and robust model of microbial population growth curves using Gaussian process (GP) regression. GP regression modeling of high-resolution time-series growth data enables accurate quantification of population growth and allows explicit control of effects from other covariates such as genetic background. This framework substantially outperforms commonly used microbial population growth models, particularly when modeling growth data from environmentally stressed populations. We apply the GP growth model and develop statistical tests to quantify the differential effects of environmental perturbations on microbial growth across a large compendium of genotypes in archaea and yeast. This method accurately identifies known transcriptional regulators and implicates novel regulators of growth under standard and stress conditions in the model archaeal organism Halobacterium salinarum For yeast, our method correctly identifies known phenotypes for a diversity of genetic backgrounds under cyclohexamide stress and also detects previously unidentified oxidative stress sensitivity across a subset of strains. Together, these results demonstrate that the GP models are interpretable, recapitulating biological knowledge of growth response while providing new insights into the relevant parameters affecting microbial population growth.
Project description:Copper (Cu) is an important enzyme co-factor that is also extremely toxic at high intracellular concentrations, making active efflux mechanisms essential for preventing Cu accumulation. Here, we have investigated the mechanistic role of metallochaperones in regulating Cu efflux. We have constructed a computational model of Cu trafficking and efflux based on systems analysis of the Cu stress response of Halobacterium salinarum. We have validated several model predictions via assays of transcriptional dynamics and intracellular Cu levels, discovering a completely novel function for metallochaperones. We demonstrate that in addition to trafficking Cu ions, metallochaperones also function as buffers to modulate the transcriptional responsiveness and efficacy of Cu efflux. This buffering function of metallochaperones ultimately sets the upper limit for intracellular Cu levels and provides a mechanistic explanation for previously observed Cu metallochaperone mutation phenotypes.