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 (Phsp5) 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 Phsp5 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 Phsp5 likely in a temperature-dependent manner. Taken together, the heat-responsiveness of Phsp5 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.
Archaea are prokaryotic organisms with simplified versions of eukaryotic transcription systems. Genes coding for the general transcription factors TBP and TFB are present in multiple copies in several Archaea, including Halobacterium sp. NRC-1. Multiple TBP and TFBs have been proposed to participate in transcription of genes via recognition and recruitment of RNA polymerase to different classes of promoters.
We attempted to knock out all six TBP and seven TFB genes in Halobacterium sp. NRC-1 using the ura3-based gene deletion system. Knockouts were obtained for six out of thirteen genes, tbpCDF and tfbACG, indicating that they are not essential for cell viability under standard conditions. Screening of a population of 1,000 candidate mutants showed that genes which did not yield mutants contained less that 0.1% knockouts, strongly suggesting that they are essential. The transcriptomes of two mutants, ΔtbpD and ΔtfbA, were compared to the parental strain and showed coordinate down regulation of many genes. Over 500 out of 2,677 total genes were regulated in the ΔtbpD and ΔtfbA mutants with 363 regulated in both, indicating that over 10% of genes in both strains require the action of both TbpD and TfbA for normal transcription. Culturing studies on the ΔtbpD and ΔtfbA mutant strains showed them to grow more slowly than the wild-type at an elevated temperature, 49°C, and they showed reduced viability at 56°C, suggesting TbpD and TfbA are involved in the heat shock response. Alignment of TBP and TFB protein sequences suggested the expansion of the TBP gene family, especially in Halobacterium sp. NRC-1, and TFB gene family in representatives of five different genera of haloarchaea in which genome sequences are available.
Six of thirteen TBP and TFB genes of Halobacterium sp. NRC-1 are non-essential under standard growth conditions. TbpD and TfbA coordinate the expression of over 10% of the genes in the NRC-1 genome. The ΔtbpD and ΔtfbA mutant strains are temperature sensitive, possibly as a result of down regulation of heat shock genes. Sequence alignments suggest the existence of several families of TBP and TFB transcription factors in Halobacterium which may function in transcription of different classes of genes.
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.
Halobacterium salinarum; Oxidative stress; Gene regulation; Transcription factor; Archaea
During evolution, enzyme-coding genes are acquired and/or replaced through lateral gene transfer and compiled into metabolic pathways. Gene regulatory networks evolve to fine tune biochemical fluxes through such metabolic pathways, enabling organisms to acclimate to nutrient fluctuations in a competitive environment. Here, we demonstrate that a single TrmB family transcription factor in Halobacterium salinarum NRC-1 globally coordinates functionally linked enzymes of diverse phylogeny in response to changes in carbon source availability. Specifically, during nutritional limitation, TrmB binds a cis-regulatory element to activate or repress 113 promoters of genes encoding enzymes in diverse metabolic pathways. By this mechanism, TrmB coordinates the expression of glycolysis, TCA cycle, and amino-acid biosynthesis pathways with the biosynthesis of their cognate cofactors (e.g. purine and thiamine). Notably, the TrmB-regulated metabolic network includes enzyme-coding genes that are uniquely archaeal as well as those that are conserved across all three domains of life. Simultaneous analysis of metabolic and gene regulatory network architectures suggests an ongoing process of co-evolution in which TrmB integrates the expression of metabolic enzyme-coding genes of diverse origins.
archaea; central metabolism; ChIP-chip; transcription regulation; TrmB
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.
archaea; ChIP–chip; non-coding RNA; tiling array; transcription
Halobacterium-halobium NRC-1 harbors a 200-kb plasmid, pNRC100, which contains a cluster of genes for synthesis of buoyant gas-filled vesicles. Physical mapping of pNRC100 by using pulsed-field gel electrophoresis showed the presence of a large (35 to 38-kb) inverted repeat (IR) sequence. Inversion isomers of pNRC100 were demonstrated by Southern hybridization analysis using two restriction enzymes, AflII and SfiI, that cut asymmetrically within the intervening small single-copy region and the large single-copy region, respectively, but not within the large IRs. No inversion isomers were observed for a deletion derivative of pNRC100 lacking one IR, which suggests that both copies are required for inversion to occur. Additionally, the identities and approximate positions of 17 insertion sequences (IS) in pNRC100 were determined by Southern hybridization and limited nucleotide sequence analysis across the IS element-target site junctions: ISH2, a 0.5-kb element, was found in four copies; ISH3, a 1.4-kb heterogeneous family of elements, was present in seven copies; ISH8, a 1.4-kb element, was found in five copies; and ISH50, a 1.0-kb element, was present in a single copy. The large IRs terminated at an ISH2 element at one end and an ISH3 element at the other end. pNRC100 is similar in structure to chloroplast and mitochondrial genomes, which contain large IRs and other large halobacterial and prokaryotic plasmids that are reservoirs of IS elements but lack the large IRs.
The halophilic archaeon Halobacterium salinarum expresses bacteriorhodopsin, a retinal-protein that allows photosynthetic growth. Transcription of the bop (bacterioopsin) gene is controlled by two transcription factors, Bat and Brz that induce bop when cells are grown anaerobically and under light.
A new gene was identified that is transcribed together with the brz gene that encodes a small basic protein designated as Brb (bacteriorhodopsin-regulating basic protein). The translation activity of the start codon of the brb gene was confirmed by BgaH reporter assays. In vivo site-directed mutagenesis of the brb gene showed that the Brb protein cooperates with Brz in the regulation of bop expression. Using a GFP reporter assay, it was demonstrated that Brb cooperates with both Brz and Bat proteins to activate bop transcription under phototrophic growth conditions.
The activation of the bop promoter was shown to be dependent not only on two major factors, Bat and Brz, but is also tuned by the small basic protein, Brb.
The genome of the halophilic archaeon Halobacterium salinarum NRC-1 encodes for homologs of MutS and MutL, which are key proteins of a DNA mismatch repair pathway conserved in Bacteria and Eukarya. Mismatch repair is essential for retaining the fidelity of genetic information and defects in this pathway result in the deleterious accumulation of mutations and in hereditary diseases in humans.
We calculated the spontaneous genomic mutation rate of H. salinarum NRC-1 using fluctuation tests targeting genes of the uracil monophosphate biosynthesis pathway. We found that H. salinarum NRC-1 has a low incidence of mutation suggesting the presence of active mechanisms to control spontaneous mutations during replication. The spectrum of mutational changes found in H. salinarum NRC-1, and in other archaea, appears to be unique to this domain of life and might be a consequence of their adaption to extreme environmental conditions. In-frame targeted gene deletions of H. salinarum NRC-1 mismatch repair genes and phenotypic characterization of the mutants demonstrated that the mutS and mutL genes are not required for maintenance of the observed mutation rate.
We established that H. salinarum NRC-1 mutS and mutL genes are redundant to an alternative system that limits spontaneous mutation in this organism. This finding leads to the puzzling question of what mechanism is responsible for maintenance of the low genomic mutation rates observed in the Archaea, which for the most part do not have MutS and MutL homologs.
Deciphering the structure of gene regulatory networks across the tree of life remains one of the major challenges in postgenomic biology. We present a novel ChIP-seq workflow for the archaea using the model organism Halobacterium salinarum sp. NRC-1 and demonstrate its application for mapping the genome-wide binding sites of natively expressed transcription factors. This end-to-end pipeline is the first protocol for ChIP-seq in archaea, with methods and tools for each stage from gene tagging to data analysis and biological discovery. Genome-wide binding sites for transcription factors with many binding sites (TfbD) are identified with sensitivity, while retaining specificity in the identification the smaller regulons (bacteriorhodopsin-activator protein). Chromosomal tagging of target proteins with a compact epitope facilitates a standardized and cost-effective workflow that is compatible with high-throughput immunoprecipitation of natively expressed transcription factors. The Pique package, an open-source bioinformatics method, is presented for identification of binding events. Relative to ChIP-Chip and qPCR, this workflow offers a robust catalog of protein–DNA binding events with improved spatial resolution and significantly decreased cost. While this study focuses on the application of ChIP-seq in H. salinarum sp. NRC-1, our workflow can also be adapted for use in other archaea and bacteria with basic genetic tools.
RNA degradation is an important factor in the regulation of gene expression. It allows organisms to quickly respond to changing environmental conditions by adapting the expression of individual genes. The stability of individual mRNAs within an organism varies considerably, contributing to differential amounts of proteins expressed. In this study we used DNA microarrays to analyze mRNA degradation in exponentially growing cultures of the extremely halophilic euryarchaeon Halobacterium salinarum NRC-1 on a global level. We determined mRNA half-lives for 1,717 open reading frames, 620 of which are part of known or predicted operons. Under the tested conditions transcript stabilities ranged from 5 min to more than 18 min, with 79% of the evaluated mRNAs showing half-lives between 8 and 12 min. The overall mean half-life was 10 min, which is considerably longer than the ones found in the other prokaryotes investigated thus far. As previously observed in Escherichia coli and Saccharomyces cerevisiae, we could not detect a significant correlation between transcript length and transcript stability, but there was a relationship between gene function and transcript stability. Genes that are known or predicted to be transcribed in operons exhibited similar mRNA half-lives. These results provide initial insights into mRNA turnover in a euryarchaeon. Moreover, our model organism, H. salinarum NRC-1, is one of just two archaea sequenced to date that are missing the core subunits of the archaeal exosome. This complex orthologous to the RNA degrading exosome of eukarya is found in all other archaeal genomes sequenced thus far.
Biogenesis of the light-driven proton pump bacteriorhodopsin in the archaeon Halobacterium salinarum requires coordinate synthesis of the bacterioopsin apoprotein and carotenoid precursors of retinal, which serves as a covalently bound cofactor. As a step towards elucidating the mechanism and regulation of carotenoid metabolism during bacteriorhodopsin biogenesis, we have identified an H. salinarum gene required for conversion of lycopene to β-carotene, a retinal precursor. The gene, designated crtY, is predicted to encode an integral membrane protein homologous to lycopene β-cyclases identified in bacteria and fungi. To test crtY function, we constructed H. salinarum strains with in-frame deletions in the gene. In the deletion strains, bacteriorhodopsin, retinal, and β-carotene were undetectable, whereas lycopene accumulated to high levels (≈1.3 nmol/mg of total cell protein). Heterologous expression of H. salinarum crtY in a lycopene-producing Escherichia coli strain resulted in β-carotene production. These results indicate that H. salinarum crtY encodes a functional lycopene β-cyclase required for bacteriorhodopsin biogenesis. Comparative sequence analysis yields a topological model of the protein and provides a plausible evolutionary connection between heterodimeric lycopene cyclases in bacteria and bifunctional lycopene cyclase-phytoene synthases in fungi.
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.
Copper (Cu) toxicity is a problem of medical, agricultural, and environmental significance. Cu toxicity severely inhibits growth of plant roots significantly affecting their morphology; Cu overload also accounts for some of the most common metal-metabolism abnormalities and neuropsychiatric problems including Wilson's and Menkes diseases. There is a large body of literature on how Cu enters and exits the cell; the kinetic and structural details of Cu translocation between trafficking, sensing, metabolic, and pumping proteins; and phenotypes associated with defects in metalloregulatory and efflux functions. Although the role of metallochaperones in Cu-cytotoxicity has been poorly studied, it has been observed that in animals deletion of metallochaperones results in elevated intracellular Cu levels along with overexpression of the P1-type ATPase efflux pump, ultimately causing malformation with high mortality. These observations are mechanistically explained by a predictive model of the Cu circuit in Halobacterium salinarum, which serves as an excellent model system for Cu trafficking and regulation in organisms with multiple chaperones. Constructed through iterative modeling and experimentation, this model accurately recapitulates known dynamical properties of the Cu circuit and predicts that intracellular Cu-buffering emerges as a consequence of the interplay of paralogous metallochaperones that traffic and allocate Cu to distinct targets.
As part of a comprehensive postgenomic investigation of the model archaeon Halobacterium sp. strain NRC-1, we used whole-genome DNA microarrays to compare transcriptional profiles of cells grown under anaerobic or aerobic conditions. When anaerobic growth supported by arginine fermentation was compared to aerobic growth, genes for arginine fermentation (arc) and anaerobic respiration (dms), using trimethylamine N-oxide (TMAO) as the terminal electron acceptor, were highly upregulated, as was the bop gene, required for phototrophic growth. When arginine fermentation was compared to anaerobic respiration with TMAO, the arc and dms genes were both induced with arginine, while TMAO induced the bop gene and major gas vesicle protein (gvpAC) genes specifying buoyant gas vesicles. Anaerobic conditions with either TMAO or arginine also upregulated the cba genes, encoding one of three cytochrome oxidases. In-frame deletion of two COG3413 family regulatory genes, bat and dmsR, showed downregulation of the bop gene cluster and loss of purple membrane synthesis and downregulation of the dms operon and loss of anaerobic respiration capability, respectively. Bioinformatic analysis identified additional regulatory and sensor genes that are likely involved in the full range of cellular responses to oxygen limitation. Our results show that the Halobacterium sp. has evolved a carefully orchestrated set of responses to oxygen limitation. As conditions become more reducing, cells progressively increase buoyancy, as well as capabilities for phototrophy, scavenging of molecular oxygen, anaerobic respiration, and fermentation.
Bacteriorhodopsin, the photosynthetic protein of Halobacterium salinarum, is optimally expressed under anaerobic growth conditions. We identified Brz (OE3104F, bacteriorhodopsin-regulating zinc finger protein), a new regulator of the bop gene. It is a small protein with a zinc finger motif, encoded directly upstream of the bop gene in the same orientation. Deletion of the brz gene caused a large decrease of bop mRNA levels as shown by Northern blot and microarray analysis. A similar effect was obtained by site-directed mutagenesis of Cys and His residues in the zinc finger motif, indicating the importance of this motif for the function of the protein. In silico analysis of the genomes from H. salinarum and other archaea revealed a large family of similar small zinc finger motif proteins, some of which may also be involved in transcription regulation of their adjacent genes.
Most studies of the transcriptional response to UV radiation in living cells have used UV doses that are much higher than those encountered in the natural environment, and most focus on short-wave UV (UV-C) at 254 nm, a wavelength that never reaches the Earth's surface. We have studied the transcriptional response of the sunlight-tolerant model archaeon, Halobacterium sp. NRC-1, to low doses of mid-wave UV (UV-B) to assess its response to UV radiation that is likely to be more biologically relevant.
Halobacterium NRC-1 cells were irradiated with UV-B at doses equivalent to 30 J/m2 and 5 J/m2 of UV-C. Transcriptional profiling showed that only 11 genes were up-regulated 1.5-fold or more by both UV-B doses. The most strongly up-regulated gene was radA1 (vng2473), the archaeal homologue of RAD51/recA recombinase. The others included arj1 (vng779) (recJ-like exonuclease), top6A (vng884) and top6B (vng885) (coding for Topoisomerase VI subunits), and nrdJ (vng1644) (which encodes a subunit of ribonucleotide reductase). We have found that four of the consistently UV-B up-regulated genes, radA1 (vng2473), vng17, top6B (vng885) and vng280, share a common 11-base pair motif in their promoter region, TTTCACTTTCA. Similar sequences were found in radA promoters in other halophilic archaea, as well as in the radA promoter of Methanospirillum hungatei. We analysed the transcriptional response of a repair-deficient ΔuvrA (vng2636) ΔuvrC (vng2381) double-deletion mutant and found common themes between it and the response in repair proficient cells.
Our results show a core set of genes is consistently up-regulated after exposure to UV-B light at low, biologically relevant doses. Eleven genes were up-regulated, in wild-type cells, after two UV-B doses (comparable to UV-C doses of 30 J/m2 and 5 J/m2), and only four genes were up-regulated by all doses of UV-B and UV-C that we have used in this work and previously. These results suggest that high doses of UV-C radiation do not necessarily provide a good model for the natural response to environmental UV. We have found an 11-base pair motif upstream of the TATA box in four of the UV-B up-regulated genes and suggest that this motif is the binding site for a transcriptional regulator involved in their response to UV damage in this model archaeon.
The bop gene of wild-type Halobacterium halobium NRC-1 is transcriptionally induced more than 20-fold under microaerobic conditions. bop transcription is inhibited by novobiocin, a DNA gyrase inhibitor, at concentrations subinhibitory for growth. The exposure of NRC-1 cultures to novobiocin concentrations inhibiting bop transcription was found to partially relax plasmid DNA supercoiling, indicating the requirement of high DNA supercoiling for bop transcription. Next, the bop promoter region was cloned on an H. halobium plasmid vector and introduced into NRC-1 and S9, a bop overproducer strain. The cloned promoter was active in both H. halobium strains, but at a higher level in the overproducer than in the wild type. Transcription from the bop promoter on the plasmid was found to be inhibited by novobiocin to a similar extent as was transcription from the chromosome. When the cloned promoter was introduced into S9 mutant strains with insertions in either of two putative regulatory genes, brp and bat, no transcription was detectable, indicating that these genes serve to activate transcription from the bop promoter in trans. Deletion analysis of the cloned bop promoter from a site approximately 480 bp upstream of bop showed that a 53-bp region 5' to the transcription start site is sufficient for transcription, but a 28-bp region is not. An 11-bp alternating purine-pyrimidine sequence within the functional promoter region, centered 23 bp 5' to the transcription start point, was found to display DNA supercoiling-dependent sensitivity to S1 nuclease and OsO4, which is consistent with a non-B-DNA conformation similar to that of left-handed Z-DNA and suggests the involvement of unusual DNA structure in supercoiling-stimulated bop gene transcription.
Proteins from extremophiles have the ability to fold and remain stable in their extreme environment. Here, we investigate the presence of this effect in the cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 (NRC-1), which was used as a model halophilic protein. The effects of salt on the structure and stability of NRC-1 and of E. coli CysRS were investigated through far-UV circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and thermal denaturation melts. The CD of NRC-1 CysRS was examined in different group I and group II chloride salts to examine the effects of the metal ions. Potassium was observed to have the strongest effect on NRC-1 CysRS structure, with the other group I salts having reduced strength. The group II salts had little effect on the protein. This suggests that the halophilic adaptations in this protein are mediated by potassium. CD and fluorescence spectra showed structural changes taking place in NRC-1 CysRS over the concentration range of 0–3 M KCl, while the structure of E. coli CysRS was relatively unaffected. Salt was also shown to increase the thermal stability of NRC-1 CysRS since the melt temperature of the CysRS from NRC-1 was increased in the presence of high salt, whereas the E. coli enzyme showed a decrease. By characterizing these interactions, this study not only explains the stability of halophilic proteins in extremes of salt, but also helps us to understand why and how group I salts stabilize proteins in general.
Gas vesicles are hollow, buoyant organelles bounded by a thin and extremely stable protein membrane. They are coded by a cluster of gvp genes in the halophilic archaeon, Halobacterium sp. NRC-1. Using an expression vector containing the entire gvp gene cluster, gas vesicle nanoparticles (GVNPs) have been successfully bioengineered for antigen display by constructing gene fusions between the gvpC gene and coding sequences from bacterial and viral pathogens.
To improve and streamline the genetic system for bioengineering of GVNPs, we first constructed a strain of Halobacterium sp. NRC-1 deleted solely for the gvpC gene. The deleted strain contained smaller, more spindle-shaped nanoparticles observable by transmission electron microscopy, confirming a shape-determining role for GvpC in gas vesicle biogenesis. Next, we constructed expression plasmids containing N-terminal coding portions or the complete gvpC gene. After introducing the expression plasmids into the Halobacterium sp. NRC-1 ΔgvpC strain, GvpC protein and variants were localized to the GVNPs by Western blotting analysis and their effects on increasing the size and shape of nanoparticles established by electron microscopy. Finally, a synthetic gene coding for Gaussia princeps luciferase was fused to the gvpC gene fragments on expression plasmids, resulting in an enzymatically active GvpC-luciferase fusion protein bound to the buoyant nanoparticles from Halobacterium.
GvpC protein and its N-terminal fragments expressed from plasmid constructs complemented a Halobacterium sp. NRC-1 ΔgvpC strain and bound to buoyant GVNPs. Fusion of the luciferase reporter gene from Gaussia princeps to the gvpC gene derivatives in expression plasmids produced GVNPs with enzymatically active luciferase bound. These results establish a significantly improved genetic system for displaying foreign proteins on Halobacterium gas vesicles and extend the bioengineering potential of these novel nanoparticles to catalytically active enzymes.
Vaccine; Halophiles; Archaea; Luciferase
By sensing changes in one or few environmental factors biological systems can anticipate future changes in multiple factors over a wide range of time scales (daily to seasonal). This anticipatory behavior is important to the fitness of diverse species, and in context of the diurnal cycle it is overall typical of eukaryotes and some photoautotrophic bacteria but is yet to be observed in archaea. Here, we report the first observation of light-dark (LD)-entrained diurnal oscillatory transcription in up to 12% of all genes of a halophilic archaeon Halobacterium salinarum NRC-1. Significantly, the diurnally entrained transcription was observed under constant darkness after removal of the LD stimulus (free-running rhythms). The memory of diurnal entrainment was also associated with the synchronization of oxic and anoxic physiologies to the LD cycle. Our results suggest that under nutrient limited conditions halophilic archaea take advantage of the causal influence of sunlight (via temperature) on O2 diffusivity in a closed hypersaline environment to streamline their physiology and operate oxically during nighttime and anoxically during daytime.
Rapidly characterizing the operational interrelationships among all genes in a given organism is a critical bottleneck to significantly advancing our understanding of thousands of newly sequenced microbial and eukaryotic species. While evolving technologies for global profiling of transcripts, proteins, and metabolites are making it possible to comprehensively survey cellular physiology in newly sequenced organisms, these experimental techniques have not kept pace with sequencing efforts. Compounding these technological challenges is the fact that individual experiments typically only stimulate relatively small-scale cellular responses, thus requiring numerous expensive experiments to survey the operational relationships among nearly all genetic elements. Therefore, a relatively quick and inexpensive strategy for observing changes in large fractions of the genetic elements is highly desirable.
We have discovered in the model organism Halobacterium salinarum NRC-1 that batch culturing in complex medium stimulates meaningful changes in the expression of approximately two thirds of all genes. While the majority of these changes occur during transition from rapid exponential growth to the stationary phase, several transient physiological states were detected beyond what has been previously observed. In sum, integrated analysis of transcript and metabolite changes has helped uncover growth phase-associated physiologies, operational interrelationships among two thirds of all genes, specialized functions for gene family members, waves of transcription factor activities, and growth phase associated cell morphology control.
Simple laboratory culturing in complex medium can be enormously informative regarding the activities of and interrelationships among a large fraction of all genes in an organism. This also yields important baseline physiological context for designing specific perturbation experiments at different phases of growth. The integration of such growth and perturbation studies with measurements of associated environmental factor changes is a practical and economical route for the elucidation of comprehensive systems-level models of biological systems.
Cellular response to stress entails complex mRNA and protein abundance changes, which translate into physiological adjustments to maintain homeostasis as well as to repair and minimize damage to cellular components. We have characterized the response of the halophilic archaeon Halobacterium salinarum NRC-1 to 60Co ionizing gamma radiation in an effort to understand the correlation between genetic information processing and physiological change. The physiological response model we have constructed is based on integrated analysis of temporal changes in global mRNA and protein abundance along with protein–DNA interactions and evolutionarily conserved functional associations. This systems view reveals cooperation among several cellular processes including DNA repair, increased protein turnover, apparent shifts in metabolism to favor nucleotide biosynthesis and an overall effort to repair oxidative damage. Further, we demonstrate the importance of time dimension while correlating mRNA and protein levels and suggest that steady-state comparisons may be misleading while assessing dynamics of genetic information processing across transcription and translation.
haloarchaea; iTRAQ; microarray; oxidative stress; proteomics
Transcription of the genomic region involved in gas vesicle formation in Halobacterium salinarum (p-vac) and Haloferax mediterranei (mc-vac) is driven by two divergent promoters, PA and PD, separated by only 35 nt. Both promoters are activated by the transcription activator GvpE which in the case of PmcA requires a 20-nt sequence (UAS) consisting of two conserved 8-nt sequence portions located upstream of BRE. Here, we determined the two UAS elements in the promoter region of p-vac by scanning mutageneses using constructs containing PpD (without PpA) fused to the bgaH reporter gene encoding an enzyme with β-galactosidase activity, or the dual reporter construct pApD with PpD fused to bgaH and PpA to an altered version of gvpA. The two UAS elements found exhibited a similar extension and distance to BRE as previously determined for the UAS in PmcA. Their distal 8-nt portions almost completely overlapped in the centre of PpD–PpA, and mutations in this region negatively affected the GvpE-mediated activation of both promoters. Any alteration of the distance between BRE and UAS resulted in the loss of the GvpE activation, as did a complete substitution of the proximal 8-nt portion, underlining that a close location of UAS and BRE was very important.
Because iron toxicity and deficiency are equally life threatening, maintaining intracellular iron levels within a narrow optimal range is critical for nearly all known organisms. However, regulatory mechanisms that establish homeostasis are not well understood in organisms that dwell in environments at the extremes of pH, temperature, and salinity. Under conditions of limited iron, the extremophile Halobacterium salinarum, a salt-loving archaeon, mounts a specific response to scavenge iron for growth. We have identified and characterized the role of two transcription factors (TFs), Idr1 and Idr2, in regulating this important response. An integrated systems analysis of TF knockout gene expression profiles and genome-wide binding locations in the presence and absence of iron has revealed that these TFs operate collaboratively to maintain iron homeostasis. In the presence of iron, Idr1 and Idr2 bind near each other at 24 loci in the genome, where they are both required to repress some genes. By contrast, Idr1 and Idr2 are both necessary to activate other genes in a putative a feed forward loop. Even at loci bound independently, the two TFs target different genes with similar functions in iron homeostasis. We discuss conserved and unique features of the Idr1–Idr2 system in the context of similar systems in organisms from other domains of life.
The Z-curve is a three-dimensional curve that
constitutes a unique representation of a DNA sequence, i.e., both the
Z-curve and the given DNA sequence can be uniquely
reconstructed from the other. We employed Z-curve
analysis to identify one replication origin in the
Methanocaldococcus jannaschii genome, two replication
origins in the Halobacterium species NRC-1 genome and
one replication origin in the Methanosarcina mazei
genome. One of the predicted replication origins of
Halobacterium species NRC-1 is the same as a
replication origin later identified by in vivo experiments. The
Z-curve analysis of the Sulfolobus
solfataricus P2 genome suggested the existence of three
replication origins, which is also consistent with later experimental
results. This review aims to summarize applications of the
Z-curve in identifying replication origins of
archaeal genomes, and to provide clues about the locations of as yet
unidentified replication origins of the Aeropyrum
pernix K1, Methanococcus maripaludis S2,
Picrophilus torridus DSM 9790 and Pyrobaculum
aerophilum str. IM2 genomes.
Halobacterium; Methanocaldococcus jannaschii; Methanosarcina mazei
Differential expression of genes can be regulated on many different levels. Most global studies of gene regulation concentrate on transcript level regulation, and very few global analyses of differential translational efficiencies exist. The studies have revealed that in Saccharomyces cerevisiae, Arabidopsis thaliana, and human cell lines translational regulation plays a significant role. Additional species have not been investigated yet. Particularly, until now no global study of translational control with any prokaryotic species was available.
A global analysis of translational control was performed with two haloarchaeal model species, Halobacterium salinarum and Haloferax volcanii. To identify differentially regulated genes, exponentially growing and stationary phase cells were compared.
More than 20% of H. salinarum transcripts are translated with non-average efficiencies. By far the largest group is comprised of genes that are translated with above-average efficiency specifically in exponential phase, including genes for many ribosomal proteins, RNA polymerase subunits, enzymes, and chemotaxis proteins. Translation of 1% of all genes is specifically repressed in either of the two growth phases. For comparison, DNA microarrays were also used to identify differential transcriptional regulation in H. salinarum, and 17% of all genes were found to have non-average transcript levels in exponential versus stationary phase.
In H. volcanii, 12% of all genes are translated with non-average efficiencies. The overlap with H. salinarum is negligible. In contrast to H. salinarum, 4.6% of genes have non-average translational efficiency in both growth phases, and thus they might be regulated by other stimuli than growth phase.
For the first time in any prokaryotic species it was shown that a significant fraction of genes is under differential translational control. Groups of genes with different regulatory patterns were discovered. However, neither the fractions nor the identity of regulated genes are conserved between H. salinarum and H. volcanii, indicating that prokaryotes as well as eukaryotes use differential translational control for the regulation of gene expression, but that the identity of regulated genes is not conserved.
For 70 H. salinarum genes potentiation of regulation was observed, but for the majority of regulated genes either transcriptional or translational regulation is employed.