SnapShot: Microbial Extremophiles.

Extremophiles are remarkable examples of life's resilience, thriving in hot springs at boiling temperatures, in brine lakes saturated with salt, and in the driest deserts. We review the biogeography, currently known limits of life, and molecular adaptations to extremes. See the online interactive map for additional detail on biogeography, environmental microbiology, and exemplary species. To view this SnapShot, open or download the PDF.

A conserved transcription factor controls gluconeogenesis via distinct targets in hypersaline-adapted archaea with diverse metabolic capabilities.

Timely regulation of carbon metabolic pathways is essential for cellular processes and to prevent futile cycling of intracellular metabolites. In Halobacterium salinarum, a hypersaline adapted archaeon, a sugar-sensing TrmB family protein controls gluconeogenesis and other biosynthetic pathways. Notably, Hbt. salinarum does not utilize carbohydrates for energy, uncommon among Haloarchaea. We characterized a TrmB-family transcriptional regulator in a saccharolytic generalist, Haloarcula hispanica, to investigate whether the targets and function of TrmB, or its regulon, is conserved in related species with distinct metabolic capabilities. In Har. hispanica, TrmB binds to 15 sites in the genome and induces the expression of genes primarily involved in gluconeogenesis and tryptophan biosynthesis. An important regulatory control point in Hbt. salinarum, activation of ppsA and repression of pykA, is absent in Har. hispanica. Contrary to its role in Hbt. salinarum and saccharolytic hyperthermophiles, TrmB does not act as a global regulator: it does not directly repress the expression of glycolytic enzymes, peripheral pathways such as cofactor biosynthesis, or catabolism of other carbon sources in Har. hispanica. Cumulatively, these findings suggest rewiring of the TrmB regulon alongside metabolic network evolution in Haloarchaea.

Halofilins as emerging bactofilin families of archaeal cell shape plasticity orchestrators.

Bactofilins are rigid, nonpolar bacterial cytoskeletal filaments that link cellular processes to specific curvatures of the cytoplasmic membrane. Although homologs of bactofilins have been identified in archaea and eukaryotes, functional studies have remained confined to bacterial systems. Here, we characterize representatives of two families of archaeal bactofilins from the pleomorphic archaeon Haloferax volcanii, halofilin A (HalA) and halofilin B (HalB). HalA and HalB polymerize in vitro, assembling into straight bundles. HalA polymers are highly dynamic and accumulate at positive membrane curvatures in vivo, whereas HalB forms more static foci that localize in areas of local negative curvatures on the outer cell surface. Gene deletions and live-cell imaging show that halofilins are critical in maintaining morphological integrity during shape transition from disk (sessile) to rod (motile). Morphological defects in ΔhalA result in accumulation of highly positive curvatures in rods but not in disks. Conversely, disk-shaped cells are exclusively affected by halB deletion, resulting in flatter cells. Furthermore, while ΔhalA and ΔhalB cells imprecisely determine the future division plane, defects arise predominantly during the disk-to-rod shape remodeling. The deletion of halA in the haloarchaeon Halobacterium salinarum, whose cells are consistently rod-shaped, impacted morphogenesis but not cell division. Increased levels of halofilins enforced drastic deformations in cells devoid of the S-layer, suggesting that HalB polymers are more stable at defective S-layer lattice regions. Our results suggest that halofilins might play a significant mechanical scaffolding role in addition to possibly directing envelope synthesis.

TroR is the primary regulator of the iron homeostasis transcription network in the halophilic archaeon Haloferax volcanii.

Maintaining the intracellular iron concentration within the homeostatic range is vital to meet cellular metabolic needs and reduce oxidative stress. Previous research revealed that the haloarchaeon Halobacterium salinarum encodes four diphtheria toxin repressor (DtxR) family transcription factors (TFs) that together regulate the iron response through an interconnected transcriptional regulatory network (TRN). However, the conservation of the TRN and the metal specificity of DtxR TFs remained poorly understood. Here we identified and characterized the TRN of Haloferax volcanii for comparison. Genetic analysis demonstrated that Hfx. volcanii relies on three DtxR transcriptional regulators (Idr, SirR, and TroR), with TroR as the primary regulator of iron homeostasis. Bioinformatics and molecular approaches revealed that TroR binds a conserved cis-regulatory motif located ∼100 nt upstream of the start codon of iron-related target genes. Transcriptomics analysis demonstrated that, under conditions of iron sufficiency, TroR repressed iron uptake and induced iron storage mechanisms. TroR repressed the expression of one other DtxR TF, Idr. This reduced DtxR TRN complexity relative to that of Hbt. salinarum appeared correlated with natural variations in iron availability. Based on these data, we hypothesize that variable environmental conditions such as iron availability appear to select for increasing TRN complexity.

TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii.

Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt-loving organism Haloferax volcanii exhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related species Halobacterium salinarum by activating the expression of enzyme-coding genes in the gluconeogenesis metabolic pathway. In Hbt. salinarum, TrmB-dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here, we use a combination of genetics and quantitative phenotyping assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions in Hfx. volcanii. The ∆trmB strain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we name trmB suppressor, or TbsP (a.k.a. "tablespoon"). TbsP is required for adhesion to abiotic surfaces (i.e., biofilm formation) and maintains wild-type cell morphology and motility. We use functional genomics and promoter fusion assays to characterize the regulons controlled by each of TrmB and TbsP, including joint regulation of the glucose-dependent transcription of gapII, which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP coregulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients in Hfx. volcanii.

Transcriptional Regulation in Archaea: From Individual Genes to Global Regulatory Networks.

Archaea are major contributors to biogeochemical cycles, possess unique metabolic capabilities, and resist extreme stress. To regulate the expression of genes encoding these unique programs, archaeal cells use gene regulatory networks (GRNs) composed of transcription factor proteins and their target genes. Recent developments in genetics, genomics, and computational methods used with archaeal model organisms have enabled the mapping and prediction of global GRN structures. Experimental tests of these predictions have revealed the dynamical function of GRNs in response to environmental variation. Here, we review recent progress made in this area, from investigating the mechanisms of transcriptional regulation of individual genes to small-scale subnetworks and genome-wide global networks. At each level, archaeal GRNs consist of a hybrid of bacterial, eukaryotic, and uniquely archaeal mechanisms. We discuss this theme from the perspective of the role of individual transcription factors in genome-wide regulation, how these proteins interact to compile GRN topological structures, and how these topologies lead to emergent, high-level GRN functions. We conclude by discussing how systems biology approaches are a fruitful avenue for addressing remaining challenges, such as discovering gene function and the evolution of GRNs.

An archaeal histone-like protein regulates gene expression in response to salt stress.

Histones, ubiquitous in eukaryotes as DNA-packing proteins, find their evolutionary origins in archaea. Unlike the characterized histone proteins of a number of methanogenic and themophilic archaea, previous research indicated that HpyA, the sole histone encoded in the model halophile Halobacterium salinarum, is not involved in DNA packaging. Instead, it was found to have widespread but subtle effects on gene expression and to maintain wild type cell morphology. However, the precise function of halophilic histone-like proteins remain unclear. Here we use quantitative phenotyping, genetics, and functional genomics to investigate HpyA function. These experiments revealed that HpyA is important for growth and rod-shaped morphology in reduced salinity. HpyA preferentially binds DNA at discrete genomic sites under low salt to regulate expression of ion uptake, particularly iron. HpyA also globally but indirectly activates other ion uptake and nucleotide biosynthesis pathways in a salt-dependent manner. Taken together, these results demonstrate an alternative function for an archaeal histone-like protein as a transcriptional regulator, with its function tuned to the physiological stressors of the hypersaline environment.

A Bayesian non-parametric mixed-effects model of microbial growth curves.

Substantive changes in gene expression, metabolism, and the proteome are manifested in overall changes in microbial population growth. Quantifying how microbes grow is therefore fundamental to areas such as genetics, bioengineering, and food safety. Traditional parametric growth curve models capture the population growth behavior through a set of summarizing parameters. However, estimation of these parameters from data is confounded by random effects such as experimental variability, batch effects or differences in experimental material. A systematic statistical method to identify and correct for such confounding effects in population growth data is not currently available. Further, our previous work has demonstrated that parametric models are insufficient to explain and predict microbial response under non-standard growth conditions. Here we develop a hierarchical Bayesian non-parametric model of population growth that identifies the latent growth behavior and response to perturbation, while simultaneously correcting for random effects in the data. This model enables more accurate estimates of the biological effect of interest, while better accounting for the uncertainty due to technical variation. Additionally, modeling hierarchical variation provides estimates of the relative impact of various confounding effects on measured population growth.

Detecting differential growth of microbial populations with Gaussian process regression.

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.

Clustering gene expression time series data using an infinite Gaussian process mixture model.

Transcriptome-wide time series expression profiling is used to characterize the cellular response to environmental perturbations. The first step to analyzing transcriptional response data is often to cluster genes with similar responses. Here, we present a nonparametric model-based method, Dirichlet process Gaussian process mixture model (DPGP), which jointly models data clusters with a Dirichlet process and temporal dependencies with Gaussian processes. We demonstrate the accuracy of DPGP in comparison to state-of-the-art approaches using hundreds of simulated data sets. To further test our method, we apply DPGP to published microarray data from a microbial model organism exposed to stress and to novel RNA-seq data from a human cell line exposed to the glucocorticoid dexamethasone. We validate our clusters by examining local transcription factor binding and histone modifications. Our results demonstrate that jointly modeling cluster number and temporal dependencies can reveal shared regulatory mechanisms. DPGP software is freely available online at https://github.com/PrincetonUniversity/DP_GP_cluster.

A transcription network of interlocking positive feedback loops maintains intracellular iron balance in archaea.

Iron is required for key metabolic processes but is toxic in excess. This circumstance forces organisms across the tree of life to tightly regulate iron homeostasis. In hypersaline lakes dominated by archaeal species, iron levels are extremely low and subject to environmental change; however, mechanisms regulating iron homeostasis in archaea remain unclear. In previous work, we demonstrated that two transcription factors (TFs), Idr1 and Idr2, collaboratively regulate aspects of iron homeostasis in the model species Halobacterium salinarum. Here we show that Idr1 and Idr2 are part of an extended regulatory network of four TFs of the bacterial DtxR family that maintains intracellular iron balance. We demonstrate that each TF directly regulates at least one of the other DtxR TFs at the level of transcription. Dynamical modeling revealed interlocking positive feedback loop architecture, which exhibits bistable or oscillatory network dynamics depending on iron availability. TF knockout mutant phenotypes are consistent with model predictions. Together, our results support that this network regulates iron homeostasis despite variation in extracellular iron levels, consistent with dynamical properties of interlocking feedback architecture in eukaryotes. These results suggest that archaea use bacterial-type TFs in a eukaryotic regulatory network topology to adapt to harsh environments.

Gene Expression of Haloferax volcanii on Intermediate and Abundant Sources of Fixed Nitrogen.

Haloferax volcanii, a well-developed model archaeon for genomic, transcriptomic, and proteomic analyses, can grow on a defined medium of abundant and intermediate levels of fixed nitrogen. Here we report a global profiling of gene expression of H. volcanii grown on ammonium as an abundant source of fixed nitrogen compared to l-alanine, the latter of which exemplifies an intermediate source of nitrogen that can be obtained from dead cells in natural habitats. By comparing the two growth conditions, 30 genes were found to be differentially expressed, including 16 genes associated with amino acid metabolism and transport. The gene expression profiles contributed to mapping ammonium and l-alanine usage with respect to transporters and metabolic pathways. In addition, conserved DNA motifs were identified in the putative promoter regions and transcription factors were found to be in synteny with the differentially expressed genes, leading us to propose regulons of transcriptionally co-regulated operons. This study provides insight to how H. volcanii responds to and utilizes intermediate vs. abundant sources of fixed nitrogen for growth, with implications for conserved functions in related halophilic archaea.

GlpR Is a Direct Transcriptional Repressor of Fructose Metabolic Genes in Haloferax volcanii.

DeoR-type helix-turn-helix (HTH) domain proteins are transcriptional regulators of sugar and nucleoside metabolism in diverse bacteria and also occur in select archaea. In the model archaeon Haloferax volcanii, previous work implicated GlpR, a DeoR-type transcriptional regulator, in the transcriptional repression of glpR and the gene encoding the fructose-specific phosphofructokinase (pfkB) during growth on glycerol. However, the global regulon governed by GlpR remained unclear. Here, we compared transcriptomes of wild-type and ΔglpR mutant strains grown on glycerol and glucose to detect significant transcript level differences for nearly 50 new genes regulated by GlpR. By coupling computational prediction of GlpR binding sequences with in vivo and in vitro DNA binding experiments, we determined that GlpR directly controls genes encoding enzymes involved in fructose degradation, including fructose bisphosphate aldolase, a central control point in glycolysis. GlpR also directly controls other transcription factors. In contrast, other metabolic pathways appear to be under the indirect influence of GlpR. In vitro experiments demonstrated that GlpR purifies to function as a tetramer that binds the effector molecule fructose-1-phosphate (F1P). These results suggest that H. volcanii GlpR functions as a direct negative regulator of fructose degradation during growth on carbon sources other than fructose, such as glucose and glycerol, and that GlpR bears striking functional similarity to bacterial DeoR-type regulators.IMPORTANCE Many archaea are extremophiles, able to thrive in habitats of extreme salinity, pH and temperature. These biological properties are ideal for applications in biotechnology. However, limited knowledge of archaeal metabolism is a bottleneck that prevents the broad use of archaea as microbial factories for industrial products. Here, we characterize how sugar uptake and use are regulated in a species that lives in high salinity. We demonstrate that a key sugar regulatory protein in this archaeal species functions using molecular mechanisms conserved with distantly related bacterial species.

A regulatory hierarchy controls the dynamic transcriptional response to extreme oxidative stress in archaea.

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.

Systems biology approaches to defining transcription regulatory networks in halophilic archaea.

To survive complex and changing environmental conditions, microorganisms use gene regulatory networks (GRNs) composed of interacting regulatory transcription factors (TFs) to control the timing and magnitude of gene expression. Genome-wide datasets; such as transcriptomics and protein-DNA interactions; and experiments such as high throughput growth curves; facilitate the construction of GRNs and provide insight into TF interactions occurring under stress. Systems biology approaches integrate these datasets into models of GRN architecture as well as statistical and/or dynamical models to understand the function of networks occurring in cells. Previously, these types of studies have focused on traditional model organisms (e.g. Escherichia coli, yeast). However, recent advances in archaeal genetics and other tools have enabled a systems approach to understanding GRNs in these relatively less studied archaeal model organisms. In this report, we outline a systems biology workflow for generating and integrating data focusing on the TF regulator. We discuss experimental design, outline the process of data collection, and provide the tools required to produce high confidence regulons for the TFs of interest. We provide a case study as an example of this workflow, describing the construction of a GRN centered on multi-TF coordinate control of gene expression governing the oxidative stress response in the hypersaline-adapted archaeon Halobacterium salinarum.

A transcription factor links growth rate and metabolism in the hypersaline adapted archaeon Halobacterium salinarum.

Co-ordinating metabolism and growth is a key challenge for all organisms. Despite fluctuating environments, cells must produce the same metabolic outputs to thrive. The mechanisms underlying this 'growth homeostasis' are known in bacteria and eukaryotes, but remain unexplored in archaea. In the model archaeon Halobacterium salinarum, the transcription factor TrmB regulates enzyme-coding genes in diverse metabolic pathways in response to glucose. However, H. salinarum is thought not to catabolize glucose. To resolve this discrepancy, we demonstrate that TrmB regulates the gluconeogenic production of sugars incorporated into the cell surface S-layer glycoprotein. Additionally, we show that TrmB-DNA binding correlates with instantaneous growth rate, likely because S-layer glycosylation is proportional to growth. This suggests that TrmB transduces a growth rate signal to co-regulated metabolic pathways including amino acid, purine, and cobalamin biosynthesis. Remarkably, the topology and function of this growth homeostatic network appear conserved across domains despite extensive alterations in protein components.

Protein-DNA binding dynamics predict transcriptional response to nutrients in archaea.

Organisms across all three domains of life use gene regulatory networks (GRNs) to integrate varied stimuli into coherent transcriptional responses to environmental pressures. However, inferring GRN topology and regulatory causality remains a central challenge in systems biology. Previous work characterized TrmB as a global metabolic transcription factor in archaeal extremophiles. However, it remains unclear how TrmB dynamically regulates its ∼100 metabolic enzyme-coding gene targets. Using a dynamic perturbation approach, we elucidate the topology of the TrmB metabolic GRN in the model archaeon Halobacterium salinarum. Clustering of dynamic gene expression patterns reveals that TrmB functions alone to regulate central metabolic enzyme-coding genes but cooperates with various regulators to control peripheral metabolic pathways. Using a dynamical model, we predict gene expression patterns for some TrmB-dependent promoters and infer secondary regulators for others. Our data suggest feed-forward gene regulatory topology for cobalamin biosynthesis. In contrast, purine biosynthesis appears to require TrmB-independent regulators. We conclude that TrmB is an important component for mediating metabolic modularity, integrating nutrient status and regulating gene expression dynamics alone and in concert with secondary regulators.

Two transcription factors are necessary for iron homeostasis in a salt-dwelling archaeon.

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 Hypersaline Archaeal Histones HpyA and HstA Are DNA Binding Proteins That Defy Categorization According to Commonly Used Functional Criteria.

Histone proteins are found across diverse lineages of Archaea, many of which package DNA and form chromatin. However, previous research has led to the hypothesis that the histone-like proteins of high-salt-adapted archaea, or halophiles, function differently. The sole histone protein encoded by the model halophilic species Halobacterium salinarum, HpyA, is nonessential and expressed at levels too low to enable genome-wide DNA packaging. Instead, HpyA mediates the transcriptional response to salt stress. Here we compare the features of genome-wide binding of HpyA to those of HstA, the sole histone of another model halophile, Haloferax volcanii. hstA, like hpyA, is a nonessential gene. To better understand HpyA and HstA functions, protein-DNA binding data (chromatin immunoprecipitation sequencing [ChIP-seq]) of these halophilic histones are compared to publicly available ChIP-seq data from DNA binding proteins across all domains of life, including transcription factors (TFs), nucleoid-associated proteins (NAPs), and histones. These analyses demonstrate that HpyA and HstA bind the genome infrequently in discrete regions, which is similar to TFs but unlike NAPs, which bind a much larger genomic fraction. However, unlike TFs that typically bind in intergenic regions, HpyA and HstA binding sites are located in both coding and intergenic regions. The genome-wide dinucleotide periodicity known to facilitate histone binding was undetectable in the genomes of both species. Instead, TF-like and histone-like binding sequence preferences were detected for HstA and HpyA, respectively. Taken together, these data suggest that halophilic archaeal histones are unlikely to facilitate genome-wide chromatin formation and that their function defies categorization as a TF, NAP, or histone. IMPORTANCE Most cells in eukaryotic species-from yeast to humans-possess histone proteins that pack and unpack DNA in response to environmental cues. These essential proteins regulate genes necessary for important cellular processes, including development and stress protection. Although the histone fold domain originated in the domain of life Archaea, the function of archaeal histone-like proteins is not well understood relative to those of eukaryotes. We recently discovered that, unlike histones of eukaryotes, histones in hypersaline-adapted archaeal species do not package DNA and can act as transcription factors (TFs) to regulate stress response gene expression. However, the function of histones across species of hypersaline-adapted archaea still remains unclear. Here, we compare hypersaline histone function to a variety of DNA binding proteins across the tree of life, revealing histone-like behavior in some respects and specific transcriptional regulatory function in others.

Comparative Analysis of rRNA Removal Methods for RNA-Seq Differential Expression in Halophilic Archaea.

Despite intense recent research interest in archaea, the scientific community has experienced a bottleneck in the study of genome-scale gene expression experiments by RNA-seq due to the lack of commercial and specifically designed rRNA depletion kits. The high rRNA:mRNA ratio (80-90%: ~10%) in prokaryotes hampers global transcriptomic analysis. Insufficient ribodepletion results in low sequence coverage of mRNA, and therefore, requires a substantially higher number of replicate samples and/or sequencing reads to achieve statistically reliable conclusions regarding the significance of differential gene expression between case and control samples. Here, we show that after the discontinuation of the previous version of RiboZero (Illumina, San Diego, CA, USA) that was useful in partially or completely depleting rRNA from archaea, archaeal transcriptomics studies have experienced a slowdown. To overcome this limitation, here, we analyze the efficiency for four different hybridization-based kits from three different commercial suppliers, each with two sets of sequence-specific probes to remove rRNA from four different species of halophilic archaea. We conclude that the key for transcriptomic success with the currently available tools is the probe-specificity for the rRNA sequence hybridization. With this paper, we provide insights into the archaeal community for selecting certain reagents and strategies over others depending on the archaeal species of interest. These methods yield improved RNA-seq sensitivity and enhanced detection of low abundance transcripts.