A Practical Guide to Small Protein Discovery and Characterization Using Mass Spectrometry.

Small proteins of up to ∼50 amino acids are an abundant class of biomolecules across all domains of life. Yet due to the challenges inherent in their size, they are often missed in genome annotations, and are difficult to identify and characterize using standard experimental approaches. Consequently, we still know few small proteins even in well-studied prokaryotic model organisms. Mass spectrometry (MS) has great potential for the discovery, validation, and functional characterization of small proteins. However, standard MS approaches are poorly suited to the identification of both known and novel small proteins due to limitations at each step of a typical proteomics workflow, i.e., sample preparation, protease digestion, liquid chromatography, MS data acquisition, and data analysis. Here, we outline the major MS-based workflows and bioinformatic pipelines used for small protein discovery and validation. Special emphasis is placed on highlighting the adjustments required to improve detection and data quality for small proteins. We discuss both the unbiased detection of small proteins and the targeted analysis of small proteins of interest. Finally, we provide guidelines to prioritize novel small proteins, and an outlook on methods with particular potential to further improve comprehensive discovery and characterization of small proteins.

Small Proteins in Archaea, a Mainly Unexplored World.

In recent years, increasing numbers of small proteins have moved into the focus of science. Small proteins have been identified and characterized in all three domains of life, but the majority remains functionally uncharacterized, lack secondary structure, and exhibit limited evolutionary conservation. While quite a few have already been described for bacteria and eukaryotic organisms, the amount of known and functionally analyzed archaeal small proteins is still very limited. In this review, we compile the current state of research, show strategies for systematic approaches for global identification of small archaeal proteins, and address selected functionally characterized examples. Besides, we document exemplarily for one archaeon the tool development and optimization to identify small proteins using genome-wide approaches.

Nitrate-Responsive Suppression of Dimethyl Sulfoxide Respiration in a Facultative Anaerobic Haloarchaeon, Haloferax volcanii.

Haloferax volcanii is a facultative anaerobic haloarchaeon that can grow using nitrate or dimethyl sulfoxide (DMSO) as a respiratory substrate under anaerobic conditions. Comparative transcriptome analysis of denitrifying and aerobic cells of H. volcanii indicated extensive changes in gene expression involving the activation of denitrification, suppression of DMSO respiration, and conversion of the heme biosynthetic pathway under denitrifying conditions. The anaerobic growth of H. volcanii by DMSO respiration was inhibited at nitrate concentrations of <1 mM, whereas nitrate-responsive growth inhibition was not observed in the ΔnarO mutant. A reporter assay demonstrated that the transcription of the dms operon was suppressed by nitrate. In contrast, the anaerobic growth of the ΔdmsR mutant by denitrification was little affected by the addition of DMSO. NarO has been identified as an activator of denitrification-related genes in response to anaerobic conditions, and here, we found that NarO is also involved in nitrate-responsive suppression of the dms operon. Nitrate-responsive suppression of DMSO respiration is known in several bacteria such as Escherichia coli and photosynthetic Rhodobacter species. This is the first report to show that a regulatory mechanism that suppresses DMSO respiration in response to nitrate exists not only in bacteria but also in haloarchaea. IMPORTANCE Haloferax volcanii can grow anaerobically by denitrification (nitrate respiration) or DMSO respiration. In facultative anaerobic bacteria that can grow by both nitrate respiration and DMSO respiration, nitrate respiration is preferentially induced when both nitrate and DMSO are available as the respiratory substrates. The results of transcriptome analysis, growth phenotyping, and reporter assays indicated that DMSO respiration is suppressed in response to nitrate in H. volcanii. The haloarchaeon-specific regulator NarO, which activates denitrification under anaerobic conditions, is suggested to be involved in the nitrate-responsive suppression of DMSO respiration.

Prokaryote toxin-antitoxin modules: Complex regulation of an unclear function.

Toxin-antitoxin (TA) modules are small operons in bacteria and archaea that encode a metabolic inhibitor (toxin) and a matching regulatory protein (antitoxin). While their biochemical activities are often well defined, their biological functions remain unclear. In Type II TA modules, the most common class, both toxin and antitoxin are proteins, and the antitoxin inhibits the biochemical activity of the toxin via complex formation with the toxin. The different TA modules vary significantly regarding structure and biochemical activity. Both regulation of protein activity by the antitoxin and regulation of transcription can be highly complex and sometimes show striking parallels between otherwise unrelated TA modules. Interplay between the multiple levels of regulation in the broader context of the cell as a whole is most likely required for optimum fine-tuning of these systems. Thus, TA modules can go through great lengths to prevent activation and to reverse accidental activation, in agreement with recent in vivo data. These complex mechanisms seem at odds with the lack of a clear biological function.

Activation of the archaeal ion channel MthK is exquisitely regulated by temperature.

Physiological response to thermal stimuli in mammals is mediated by a structurally diverse class of ion channels, many of which exhibit polymodal behavior. To probe the diversity of biophysical mechanisms of temperature-sensitivity, we characterized the temperature-dependent activation of MthK, a two transmembrane calcium-activated potassium channel from thermophilic archaebacteria. Our functional complementation studies show that these channels are more efficient at rescuing K+ transport at 37°C than at 24°C. Electrophysiological activity of the purified MthK is extremely sensitive (Q10 >100) to heating particularly at low-calcium concentrations whereas channels lacking the calcium-sensing RCK domain are practically insensitive. By analyzing single-channel activities at limiting calcium concentrations, we find that temperature alters the coupling between the cytoplasmic RCK domains and the pore domain. These findings reveal a hitherto unexplored mechanism of temperature-dependent regulation of ion channel gating and shed light on ancient origins of temperature-sensitivity.

Dual Host-Intracellular Parasite Transcriptome of Enucleated Cells Hosting Leishmania amazonensis: Control of Half-Life of Host Cell Transcripts by the Parasite.

Enucleated cells or cytoplasts (cells whose nucleus is removed in vitro) represent an unexplored biological model for intracellular infection studies due to the abrupt interruption of nuclear processing and new RNA synthesis by the host cell in response to pathogen entry. Using enucleated fibroblasts hosting the protozoan parasite Leishmania amazonensis, we demonstrate that parasite multiplication and biogenesis of large parasitophorous vacuoles in which parasites multiply are independent of the host cell nucleus. Dual RNA sequencing of both host cytoplast and intracellular parasite transcripts identified host transcripts that are more preserved or degraded upon interaction with parasites and also parasite genes that are differentially expressed when hosted by nucleated or enucleated cells. Cytoplasts are suitable host cells, which persist in culture for more than 72 h and display functional enrichment of transcripts related to mitochondrial functions and mRNA translation. Crosstalk between nucleated host de novo gene expression in response to intracellular parasitism and the parasite gene expression to counteract or benefit from these host responses induces a parasite transcriptional profile favoring parasite multiplication and aerobic respiration, and a host-parasite transcriptional landscape enriched in host cell metabolic functions related to NAD, fatty acid, and glycolytic metabolism. Conversely, interruption of host nucleus-parasite cross talk by infection of enucleated cells generates a host-parasite transcriptional landscape in which cytoplast transcripts are enriched in phagolysosome-related pathway, prosurvival, and SerpinB-mediated immunomodulation. In addition, predictive in silico analyses indicated that parasite transcript products secreted within cytoplasts interact with host transcript products conserving the host V-ATPase proton translocation function and glutamine/proline metabolism. The collective evidence indicates parasite-mediated control of host cell transcripts half-life that is beneficial to parasite intracellular multiplication and escape from host immune responses. These findings will contribute to improved drug targeting and serve as database for L. amazonensis-host cell interactions.

Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants.

The number and diversity of known CRISPR-Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR-Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR-Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR-Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR-Cas.

Structure and function of archaeal histones.

The genomes of all organisms throughout the tree of life are compacted and organized in chromatin by association of chromatin proteins. Eukaryotic genomes encode histones, which are assembled on the genome into octamers, yielding nucleosomes. Post-translational modifications of the histones, which occur mostly on their N-terminal tails, define the functional state of chromatin. Like eukaryotes, most archaeal genomes encode histones, which are believed to be involved in the compaction and organization of their genomes. Instead of discrete multimers, in vivo data suggest assembly of "nucleosomes" of variable size, consisting of multiples of dimers, which are able to induce repression of transcription. Based on these data and a model derived from X-ray crystallography, it was recently proposed that archaeal histones assemble on DNA into "endless" hypernucleosomes. In this review, we discuss the amino acid determinants of hypernucleosome formation and highlight differences with the canonical eukaryotic octamer. We identify archaeal histones differing from the consensus, which are expected to be unable to assemble into hypernucleosomes. Finally, we identify atypical archaeal histones with short N- or C-terminal extensions and C-terminal tails similar to the tails of eukaryotic histones, which are subject to post-translational modification. Based on the expected characteristics of these archaeal histones, we discuss possibilities of involvement of histones in archaeal transcription regulation.

Leaderless mRNAs in the Spotlight: Ancient but Not Outdated!

Previously, leaderless mRNAs (lmRNAs) were perceived to make up only a minor fraction of the transcriptome in bacteria. However, advancements in RNA sequencing technology are uncovering vast numbers of lmRNAs, particularly in archaea, Actinobacteria, and extremophiles and thus underline their significance in cellular physiology and regulation. Due to the absence of conventional ribosome binding signals, lmRNA translation initiation is distinct from canonical mRNAs and can therefore be differentially regulated. The ribosome's inherent ability to bind a 5'-terminal AUG can stabilize and protect the lmRNA from degradation or allow ribosomal loading for downstream initiation events. As a result, lmRNAs remain translationally competent during a variety of physiological conditions, allowing them to contribute to multiple regulatory mechanisms. Furthermore, the abundance of lmRNAs can increase during adverse conditions through the upregulation of lmRNA transcription from alternative promoters or by the generation of lmRNAs from canonical mRNAs cleaved by an endonucleolytic toxin. In these ways, lmRNA translation can continue during stress and contribute to regulation, illustrating their importance in the cell. Due to their presence in all domains of life and their ability to be translated by heterologous hosts, lmRNAs appear further to represent ancestral transcripts that might allow us to study the evolution of the ribosome and the translational process.

Patterns of thaumarchaeal gene expression in culture and diverse marine environments.

Thaumarchaea are ubiquitous in marine habitats where they participate in carbon and nitrogen cycling. Although metatranscriptomes suggest thaumarchaea are active microbes in marine waters, we understand little about how thaumarchaeal gene expression patterns relate to substrate utilization and activity. Here, we report the global transcriptional response of the marine ammonia-oxidizing thaumarchaeon 'Candidatus Nitrosopelagicus brevis' str. CN25 to ammonia limitation using RNA-Seq. We further describe the genome and transcriptome of Ca. N. brevis str. U25, a new strain capable of urea utilization. Ammonia limitation in CN25 resulted in reduced expression of transcripts coding for ammonia oxidation proteins, and increased expression of a gene coding an Hsp20-like chaperone. Despite significantly different transcript abundances across treatments, two ammonia monooxygenase subunits (amoAB), a nitrite reductase (nirK) and both ammonium transporter genes were always among the most abundant transcripts, regardless of growth state. Ca. N. brevis str. U25 cells expressed a urea transporter 139-fold more than the urease catalytic subunit ureC. Gene coexpression networks derived from culture transcriptomes and 10 thaumarchaea-enriched metatranscriptomes revealed a high degree of correlated gene expression across disparate environmental conditions and identified a module of coexpressed genes, including amoABC and nirK, that we hypothesize to represent the core ammonia oxidation machinery.

Enzymatic reconstitution of ribosomal peptide backbone thioamidation.

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.

Gene expression and ultrastructure of meso- and thermophilic methanotrophic consortia.

The sulfate-dependent, anaerobic oxidation of methane (AOM) is an important sink for methane in marine environments. It is carried out between anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) living in syntrophic partnership. In this study, we compared the genomes, gene expression patterns and ultrastructures of three phylogenetically different microbial consortia found in hydrocarbon-rich environments under different temperature regimes: ANME-1a/HotSeep-1 (60°C), ANME-1a/Seep-SRB2 (37°C) and ANME-2c/Seep-SRB2 (20°C). All three ANME encode a reverse methanogenesis pathway: ANME-2c encodes all enzymes, while ANME-1a lacks the gene for N5,N10-methylene tetrahydromethanopterin reductase (mer) and encodes a methylenetetrahydrofolate reductase (Met). The bacterial partners contain the genes encoding the canonical dissimilatory sulfate reduction pathway. During AOM, all three consortia types highly expressed genes encoding for the formation of flagella or type IV pili and/or c-type cytochromes, some predicted to be extracellular. ANME-2c expressed potentially extracellular cytochromes with up to 32 hemes, whereas ANME-1a and SRB expressed less complex cytochromes (≤ 8 and ≤ 12 heme respectively). The intercellular space of all consortia showed nanowire-like structures and heme-rich areas. These features are proposed to enable interspecies electron exchange, hence suggesting that direct electron transfer is a common mechanism to sulfate-dependent AOM, and that both partners synthesize molecules to enable it.

Formate hydrogenlyase and formate secretion ameliorate H2 inhibition in the hyperthermophilic archaeon Thermococcus paralvinellae.

Some hyperthermophilic heterotrophs in the genus Thermococcus produce H2 in the absence of S° and have up to seven hydrogenases, but their combined physiological roles are unclear. Here, we show which hydrogenases in Thermococcus paralvinellae are affected by added H2 during growth without S°. Growth rates and steady-state cell concentrations decreased while formate production rates increased when T. paralvinallae was grown in a chemostat with 65 µM of added H2(aq) . Differential gene expression analysis using RNA-Seq showed consistent expression of six hydrogenase operons with and without added H2 . In contrast, expression of the formate hydrogenlyase 1 (fhl1) operon increased with added H2 . Flux balance analysis showed H2 oxidation and formate production using FHL became an alternate route for electron disposal during H2 inhibition with a concomitant increase in growth rate relative to cells without FHL. T. paralvinellae also grew on formate with an increase in H2 production rate relative to growth on maltose or tryptone. Growth on formate increased fhl1 expression but decreased expression of all other hydrogenases. Therefore, Thermococcus that possess fhl1 have a competitive advantage over other Thermococcus species in hot subsurface environments where organic substrates are present, S° is absent and slow H2 efflux causes growth inhibition.

ThiN as a Versatile Domain of Transcriptional Repressors and Catalytic Enzymes of Thiamine Biosynthesis.

Thiamine biosynthesis is commonly regulated by a riboswitch mechanism; however, the enzymatic steps and regulation of this pathway in archaea are poorly understood. Haloferax volcanii, one of the representative archaea, uses a eukaryote-like Thi4 (thiamine thiazole synthase) for the production of the thiazole ring and condenses this ring with a pyrimidine moiety synthesized by an apparent bacterium-like ThiC (2-methyl-4-amino-5-hydroxymethylpyrimidine [HMP] phosphate synthase) branch. Here we found that archaeal Thi4 and ThiC were encoded by leaderless transcripts, ruling out a riboswitch mechanism. Instead, a novel ThiR transcription factor that harbored an N-terminal helix-turn-helix (HTH) DNA binding domain and C-terminal ThiN (TMP synthase) domain was identified. In the presence of thiamine, ThiR was found to repress the expression of thi4 and thiC by a DNA operator sequence that was conserved across archaeal phyla. Despite having a ThiN domain, ThiR was found to be catalytically inactive in compensating for the loss of ThiE (TMP synthase) function. In contrast, bifunctional ThiDN, in which the ThiN domain is fused to an N-terminal ThiD (HMP/HMP phosphate [HMP-P] kinase) domain, was found to be interchangeable for ThiE function and, thus, active in thiamine biosynthesis. A conserved Met residue of an extended α-helix near the active-site His of the ThiN domain was found to be important for ThiDN catalytic activity, whereas the corresponding Met residue was absent and the α-helix was shorter in ThiR homologs. Thus, we provide new insight into residues that distinguish catalytic from noncatalytic ThiN domains and reveal that thiamine biosynthesis in archaea is regulated by a transcriptional repressor, ThiR, and not by a riboswitch.IMPORTANCE Thiamine pyrophosphate (TPP) is a cofactor needed for the enzymatic activity of many cellular processes, including central metabolism. In archaea, thiamine biosynthesis is an apparent chimera of eukaryote- and bacterium-type pathways that is not well defined at the level of enzymatic steps or regulatory mechanisms. Here we find that ThiN is a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis in archaea. Our study provides new insight into residues that distinguish catalytic from noncatalytic ThiN domains and reveals that archaeal thiamine biosynthesis is regulated by a ThiN domain transcriptional repressor, ThiR, and not by a riboswitch.

ArtA-Dependent Processing of a Tat Substrate Containing a Conserved Tripartite Structure That Is Not Localized at the C Terminus.

Most prokaryote-secreted proteins are transported to the cell surface using either the general secretion (Sec) or twin-arginine translocation (Tat) pathway. A majority of secreted proteins are anchored to the cell surface, while the remainder are released into the extracellular environment. The anchored surface proteins play a variety of important roles in cellular processes, ranging from facilitating interactions between cells to maintaining cell stability. The extensively studied S-layer glycoprotein (SLG) of Haloferax volcanii, previously thought to be anchored via C-terminal intercalation into the membrane, was recently shown to be lipidated and to have its C-terminal segment removed in processes dependent upon archaeosortase A (ArtA), a recently discovered enzyme. While SLG is a Sec substrate, in silico analyses presented here reveal that, of eight additional ArtA substrates predicted, two substrates also contain predicted Tat signal peptides, including Hvo_0405, which has a highly conserved tripartite structure that lies closer to the center of the protein than to its C terminus, unlike other predicted ArtA substrates identified to date. We demonstrate that, even given its atypical location, this tripartite structure, which likely resulted from the fusion of genes encoding an ArtA substrate and a cytoplasmic protein, is processed in an ArtA-dependent manner. Using an Hvo_0405 mutant lacking the conserved "twin" arginines of the predicted Tat signal peptide, we show that Hvo_0405 is indeed a Tat substrate and that ArtA substrates include both Sec and Tat substrates. Finally, we confirmed the Tat-dependent localization and signal peptidase I (SPase I) cleavage site of Hvo_0405 using mass spectrometry.IMPORTANCE The specific mechanisms that facilitate protein anchoring to the archaeal cell surface remain poorly understood. Here, we have shown that the proteins bound to the cell surface of the model archaeon H. volcanii, through a recently discovered novel ArtA-dependent anchoring mechanism, are more structurally diverse than was previously known. Specifically, our results demonstrate that both Tat and Sec substrates, which contain the conserved tripartite structure of predicted ArtA substrates, can be processed in an ArtA-dependent manner and that the tripartite structure need not lie near the C terminus for this processing to occur. These data improve our understanding of archaeal cell biology and are invaluable for in silico subcellular localization predictions of archaeal and bacterial proteins.

Functional Role of MrpA in the MrpABCDEFG Na+/H+ Antiporter Complex from the Archaeon Methanosarcina acetivorans.

The multisubunit cation/proton antiporter 3 family, also called Mrp, is widely distributed in all three phylogenetic domains (Eukarya, Bacteria, and Archaea). Investigations have focused on Mrp complexes from the domain Bacteria to the exclusion of Archaea, with a consensus emerging that all seven subunits are required for Na+/H+ antiport activity. The MrpA subunit from the MrpABCDEFG Na+/H+ antiporter complex of the archaeon Methanosarcina acetivorans was produced in antiporter-deficient Escherichia coli strains EP432 and KNabc and biochemically characterized to determine the role of MrpA in the complex. Both strains containing MrpA grew in the presence of up to 500 mM NaCl and pH values up to 11.0 with no added NaCl. Everted vesicles from the strains containing MrpA were able to generate a NADH-dependent pH gradient (ΔpH), which was abated by the addition of monovalent cations. The apparent Km values for Na+ and Li+ were similar and ranged from 31 to 63 mM, whereas activity was too low to determine the apparent Km for K+ Optimum activity was obtained between pH 7.0 and 8.0. Homology molecular modeling identified two half-closed symmetry-related ion translocation channels that are linked, forming a continuous path from the cytoplasm to the periplasm, analogous to the NuoL subunit of complex I. Bioinformatics analyses revealed genes encoding homologs of MrpABCDEFG in metabolically diverse methane-producing species. Overall, the results advance the biochemical, evolutionary, and physiological understanding of Mrp complexes that extends to the domain Archaea IMPORTANCE: The work is the first reported characterization of an Mrp complex from the domain Archaea, specifically methanogens, for which Mrp is important for acetotrophic growth. The results show that the MrpA subunit is essential for antiport activity and, importantly, that not all seven subunits are required, which challenges current dogma for Mrp complexes from the domain Bacteria A mechanism is proposed in which an MrpAD subcomplex catalyzes Na+/H+ antiport independent of an MrpBCEFG subcomplex, although the activity of the former is modulated by the latter. Properties of MrpA strengthen proposals that the Mrp complex is of ancient origin and that subunits were recruited to evolve the ancestral complex I. Finally, bioinformatics analyses indicate that Mrp complexes function in diverse methanogenic pathways.

A Novel Transcriptional Regulator Related to Thiamine Phosphate Synthase Controls Thiamine Metabolism Genes in Archaea.

Thiamine (vitamin B1) is a precursor of thiamine pyrophosphate (TPP), an essential coenzyme in the central metabolism of all living organisms. Bacterial thiamine biosynthesis and salvage genes are controlled at the RNA level by TPP-responsive riboswitches. In Archaea, TPP riboswitches are restricted to the Thermoplasmatales order. Mechanisms of transcriptional control of thiamine genes in other archaeal lineages remain unknown. Using the comparative genomics approach, we identified a novel family of transcriptional regulators (named ThiR) controlling thiamine biosynthesis and transport genes in diverse lineages in the Crenarchaeota phylum as well as in the Halobacteria and Thermococci classes of the Euryarchaeota ThiR regulators are composed of an N-terminal DNA-binding domain and a C-terminal ligand-binding domain, which is similar to the archaeal thiamine phosphate synthase ThiN. By using comparative genomics, we predicted ThiR-binding DNA motifs and reconstructed ThiR regulons in 67 genomes representing all above-mentioned lineages. The predicted ThiR-binding motifs are characterized by palindromic symmetry with several distinct lineage-specific consensus sequences. In addition to thiamine biosynthesis genes, the reconstructed ThiR regulons include various transporters for thiamine and its precursors. Bioinformatics predictions were experimentally validated by in vitro DNA-binding assays with the recombinant ThiR protein from the hyperthermophilic archaeon Metallosphaera yellowstonensis MK1. Thiamine phosphate and, to some extent, TPP and hydroxyethylthiazole phosphate were required for the binding of ThiR to its DNA targets, suggesting that ThiR is derepressed by limitation of thiamine phosphates. The thiamine phosphate-binding residues previously identified in ThiN are highly conserved in ThiR regulators, suggesting a conserved mechanism for effector recognition. IMPORTANCE: Thiamine pyrophosphate is a cofactor for many essential enzymes for glucose and energy metabolism. Thiamine or vitamin B1 biosynthesis and its transcriptional regulation in Archaea are poorly understood. We applied the comparative genomics approach to identify a novel family of regulators for the transcriptional control of thiamine metabolism genes in Archaea and reconstructed the respective regulons. The predicted ThiR regulons in archaeal genomes control the majority of thiamine biosynthesis genes. The reconstructed regulon content suggests that numerous uptake transporters for thiamine and/or its precursors are encoded in archaeal genomes. The ThiR regulon was experimentally validated by DNA-binding assays with Metallosphaera spp. These discoveries contribute to our understanding of metabolic and regulatory networks involved in vitamin homeostasis in diverse lineages of Archaea.

Malate Synthase and ß-Methylmalyl Coenzyme A Lyase Reactions in the Methylaspartate Cycle in Haloarcula hispanica.

Haloarchaea are extremely halophilic heterotrophic microorganisms belonging to the class Halobacteria (Euryarchaeota). Almost half of the haloarchaea possesses the genes coding for enzymes of the methylaspartate cycle, a recently discovered anaplerotic acetate assimilation pathway. In this cycle, the enzymes of the tricarboxylic acid cycle together with the dedicated enzymes of the methylaspartate cycle convert two acetyl coenzyme A (acetyl-CoA) molecules to malate. The methylaspartate cycle involves two reactions catalyzed by homologous enzymes belonging to the CitE-like enzyme superfamily, malyl-CoA lyase/thioesterase (haloarchaeal malate synthase [hMS]; Hah_2476 in Haloarcula hispanica) and ß-methylmalyl-CoA lyase (haloarchaeal ß-methylmalyl-CoA lyase [hMCL]; Hah_1341). Although both enzymes catalyze the same reactions, hMS was previously proposed to preferentially catalyze the formation of malate from acetyl-CoA and glyoxylate (malate synthase activity) and hMCL was proposed to primarily cleave ß-methylmalyl-CoA to propionyl-CoA and glyoxylate. Here we studied the physiological functions of these enzymes during acetate assimilation in H. hispanica by using biochemical assays of the wild type and deletion mutants. Our results reveal that the main physiological function of hMS is malyl-CoA (not malate) formation and that hMCL catalyzes a ß-methylmalyl-CoA lyase reaction in vivo The malyl-CoA thioesterase activities of both enzymes appear to be not essential for growth on acetate. Interestingly, despite the different physiological functions of hMS and hMCL, structural comparisons predict that these two proteins have virtually identical active sites, thus highlighting the need for experimental validation of their catalytic functions. Our results provide further proof of the operation of the methylaspartate cycle and indicate the existence of a distinct, yet-to-be-discovered malyl-CoA thioesterase in haloarchaea. IMPORTANCE: Acetate is one of the most important substances in natural environments. The activated form of acetate, acetyl coenzyme A (acetyl-CoA), is the high-energy intermediate at the crossroads of central metabolism: its oxidation generates energy for the cell, and about a third of all biosynthetic fluxes start directly from acetyl-CoA. Many organic compounds enter the central carbon metabolism via this key molecule. To sustain growth on acetyl-CoA-generating compounds, a dedicated assimilation (anaplerotic) pathway is required. The presence of an anaplerotic pathway is a prerequisite for growth in many environments, being important for environmentally, industrially, and clinically important microorganisms. Here we studied specific reactions of a recently discovered acetate assimilation pathway, the methylaspartate cycle, functioning in extremely halophilic archaea.

An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism.

Routes for cysteine biosynthesis are still unknown in many archaea. Here we find that the hyperthermophilic archaeon Thermococcus kodakarensis generates cysteine from serine via O-phosphoserine, in addition to the classical route from 3-phosphoglycerate. The protein responsible for serine phosphorylation is encoded by TK0378, annotated as a chromosome partitioning protein ParB. The TK0378 protein utilizes ADP as the phosphate donor, but in contrast to previously reported ADP-dependent kinases, recognizes a non-sugar substrate. Activity is specific towards free serine, and not observed with threonine, homoserine and serine residues within a peptide. Genetic analyses suggest that TK0378 is involved in serine assimilation and clearly responsible for cysteine biosynthesis from serine. TK0378 homologs, present in Thermococcales and Desulfurococcales, are most likely not ParB proteins and constitute a group of kinases involved in serine utilization.

Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase.

UNLABELLED: The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcanii IMPORTANCE: The work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii The functional involvement of the three enzymes was proven by analyses of the corresponding knockout mutants. These results provide evidence for the in vivo operation of the semiphosphorylative Entner-Doudoroff pathway in haloarchaea and thus expand our understanding of the unusual sugar degradation pathways in the domain Archaea.