Regulation In Archaea Research Articles

Chromatin and gene regulation in archaea.

The chromatinisation of DNA by nucleoid-associated proteins (NAPs) in archaea 'formats' the genome structure in profound ways, revealing both striking differences and analogies to eukaryotic chromatin. However, the extent to which archaeal NAPs actively regulate gene expression remains poorly understood. The dawn of quantitative chromatin mapping techniques and first NAP-specific occupancy profiles in different archaea promise a more accurate view. A picture emerges where in diverse archaea with very different NAP repertoires chromatin maintains access to regulatory motifs including the gene promoter independently of transcription activity. Our re-analysis of genome-wide occupancy data of the crenarchaeal NAP Cren7 shows that these chromatin-free regions are flanked by increased Cren7 binding across the transcription start site. While bacterial NAPs often form heterochromatin-like regions across islands with xenogeneic genes that are transcriptionally silenced, there is little evidence for similar structures in archaea and data from Haloferax show that the promoters of xenogeneic genes remain accessible. Local changes in chromatinisation causing wide-ranging effects on transcription restricted to one chromosomal interaction domain (CID) in Saccharolobus islandicus hint at a higher-order level of organisation between chromatin and transcription. The emerging challenge is to integrate results obtained at microscale and macroscale, reconciling molecular structure and function with dynamic genome-wide chromatin landscapes.

In Vitro Transcription Assay for Archaea Belonging to Sulfolobales.

Archaeal transcription and its regulation are characterized by a mosaic of eukaryotic and bacterial features. Molecular analysis of the functioning of the archaeal RNA polymerase, basal transcription factors, and specific promoter-containing DNA templates allows to unravel the mechanisms of transcription regulation in archaea. In vitro transcription is a technique that allows the study of this process in a simplified and controlled environment less complex than the archaeal cell. In this chapter, we present an in vitro transcription methodology for the study of transcription in Sulfolobales. It is described how to purify the RNA polymerase and the basal transcription factors TATA-binding protein and transcription factor B of Saccharolobus solfataricus and how to perform in vitro transcription reactions and transcript detection. Application of this protocol for other archaeal species could require minor modifications to protein overexpression and purification conditions.

The TK0271 Protein Activates Transcription of Aromatic Amino Acid Biosynthesis Genes in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis (trp) operon. Here, we show that the TrpY homolog in Thermococcus kodakarensis is not involved in such regulation. There are 87 genes on the T. kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. kodakarensis, we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli, and the protein, here designated Tar ( Thermococcalesaromatic amino acid regulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant (Kd ) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis (tyr-phe) and aro operons. The protein recognized a palindromic sequence (TGGACA-N8-TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. kodakarensis in amino acid-based medium and found that transcript levels of the trp, tyr-phe, and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. kodakarensisIMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage.

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.

RIP-Seq Suggests Translational Regulation by L7Ae in Archaea.

ABSTRACTL7Ae is a universal archaeal protein that recognizes and stabilizes kink-turn (k-turn) motifs in RNA substrates. These structural motifs are widespread in nature and are found in many functional RNA species, including ribosomal RNAs. Synthetic biology approaches utilize L7Ae/k-turn interactions to control gene expression in eukaryotes. Here, we present results of comprehensive RNA immunoprecipitation sequencing (RIP-Seq) analysis of genomically tagged L7Ae from the hyperthermophilic archaeon Sulfolobus acidocaldarius. A large set of interacting noncoding RNAs was identified. In addition, several mRNAs, including the l7ae transcript, were found to contain k-turn motifs that facilitate L7Ae binding. In vivo studies showed that L7Ae autoregulates the translation of its mRNA by binding to a k-turn motif present in the 5′ untranslated region (UTR). A green fluorescent protein (GFP) reporter system was established in Escherichia coli and verified conservation of L7Ae-mediated feedback regulation in Archaea. Mobility shift assays confirmed binding to a k-turn in the transcript of nop5-fibrillarin, suggesting that the expression of all C/D box sRNP core proteins is regulated by L7Ae. These studies revealed that L7Ae-mediated gene regulation evolved in archaeal organisms, generating new tools for the modulation of synthetic gene circuits in bacteria.

Transcription Regulation in Archaea.

The known diversity of metabolic strategies and physiological adaptations of archaeal species to extreme environments is extraordinary. Accurate and responsive mechanisms to ensure that gene expression patterns match the needs of the cell necessitate regulatory strategies that control the activities and output of the archaeal transcription apparatus. Archaea are reliant on a single RNA polymerase for all transcription, and many of the known regulatory mechanisms employed for archaeal transcription mimic strategies also employed for eukaryotic and bacterial species. Novel mechanisms of transcription regulation have become apparent by increasingly sophisticated in vivo and in vitro investigations of archaeal species. This review emphasizes recent progress in understanding archaeal transcription regulatory mechanisms and highlights insights gained from studies of the influence of archaeal chromatin on transcription.

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

The metabolism of Archaea, the third domain of life, resembles in its complexity those of Bacteria and lower Eukarya. However, this metabolic complexity in Archaea is accompanied by the absence of many "classical" pathways, particularly in central carbohydrate metabolism. Instead, Archaea are characterized by the presence of unique, modified variants of classical pathways such as the Embden-Meyerhof-Parnas (EMP) pathway and the Entner-Doudoroff (ED) pathway. The pentose phosphate pathway is only partly present (if at all), and pentose degradation also significantly differs from that known for bacterial model organisms. These modifications are accompanied by the invention of "new," unusual enzymes which cause fundamental consequences for the underlying regulatory principles, and classical allosteric regulation sites well established in Bacteria and Eukarya are lost. The aim of this review is to present the current understanding of central carbohydrate metabolic pathways and their regulation in Archaea. In order to give an overview of their complexity, pathway modifications are discussed with respect to unusual archaeal biocatalysts, their structural and mechanistic characteristics, and their regulatory properties in comparison to their classic counterparts from Bacteria and Eukarya. Furthermore, an overview focusing on hexose metabolic, i.e., glycolytic as well as gluconeogenic, pathways identified in archaeal model organisms is given. Their energy gain is discussed, and new insights into different levels of regulation that have been observed so far, including the transcript and protein levels (e.g., gene regulation, known transcription regulators, and posttranslational modification via reversible protein phosphorylation), are presented.

The First Prokaryotic Trehalose Synthase Complex Identified in the Hyperthermophilic Crenarchaeon Thermoproteus tenax

The role of the disaccharide trehalose, its biosynthesis pathways and their regulation in Archaea are still ambiguous. In Thermoproteus tenax a fused trehalose-6-phosphate synthase/phosphatase (TPSP), consisting of an N-terminal trehalose-6-phosphate synthase (TPS) and a C-terminal trehalose-6-phosphate phosphatase (TPP) domain, was identified. The tpsp gene is organized in an operon with a putative glycosyltransferase (GT) and a putative mechanosensitive channel (MSC). The T. tenax TPSP exhibits high phosphatase activity, but requires activation by the co-expressed GT for bifunctional synthase-phosphatase activity. The GT mediated activation of TPS activity relies on the fusion of both, TPS and TPP domain, in the TPSP enzyme. Activation is mediated by complex-formation in vivo as indicated by yeast two-hybrid and crude extract analysis. In combination with first evidence for MSC activity the results suggest a sophisticated stress response involving TPSP, GT and MSC in T. tenax and probably in other Thermoproteales species. The monophyletic prokaryotic TPSP proteins likely originated via a single fusion event in the Bacteroidetes with subsequent horizontal gene transfers to other Bacteria and Archaea. Furthermore, evidence for the origin of eukaryotic TPSP fusions via HGT from prokaryotes and therefore a monophyletic origin of eukaryotic and prokaryotic fused TPSPs is presented. This is the first report of a prokaryotic, archaeal trehalose synthase complex exhibiting a much more simple composition than the eukaryotic complex described in yeast. Thus, complex formation and a complex-associated regulatory potential might represent a more general feature of trehalose synthesizing proteins.

Small RNAs for defence and regulation in archaea

Non-coding RNAs are key players in many cellular processes within organisms from all three domains of life. The range and diversity of small RNA functions beyond their involvement in translation and RNA processing was first recognized for eukaryotes and bacteria. Since then, small RNAs were also found to be abundant in archaea. Their functions include the regulation of gene expression and the establishment of immunity against invading mobile genetic elements. This review summarizes our current knowledge about small RNAs used for regulation and defence in archaea.

Growth phase-dependent gene regulation in vivo in Sulfolobus solfataricus

Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene regulation in Archaea. SSV1-based lacS reporter gene constructs containing the Sulfolobus 16S/23S rRNA gene core promoter, the TF55α core promoter, or the native lacS promoter were tested in Sulfolobus solfataricus cells lacking the lacS gene. The 42-bp 16S/23S rRNA gene and 39-bp TF55α core promoters are sufficient for gene expression in S. solfataricus. However, only gene expression driven by the 16S/23S rRNA gene core promoter is dependent on the culture growth phase. This is the smallest known regulated promoter in Sulfolobus. To our knowledge, this is the first study to show growth phase-dependent rRNA gene regulation in Archaea.

Regulation of Translation in Haloarchaea: 5′- and 3′-UTRs Are Essential and Have to Functionally Interact In Vivo

Recently a first genome-wide analysis of translational regulation using prokaryotic species had been performed which revealed that regulation of translational efficiency plays an important role in haloarchaea. In fact, the fractions of genes under differential growth phase-dependent translational control in the two species Halobacterium salinarum and Haloferax volcanii were as high as in eukaryotes. However, nothing is known about the mechanisms of translational regulation in archaea. Therefore, two genes exhibiting opposing directions of regulation were selected to unravel the importance of untranslated regions (UTRs) for differential translational control in vivo.Differential translational regulation in exponentially growing versus stationary phase cells was studied by comparing translational efficiencies using a reporter gene system. Translational regulation was not observed when 5′-UTRs or 3′-UTRs alone were fused to the reporter gene. However, their simultaneous presence was sufficient to transfer differential translational control from the native transcript to the reporter transcript. This was true for both directions of translational control. Translational regulation was completely abolished when stem loops in the 5′-UTR were changed by mutagenesis. An “UTR-swap” experiment demonstrated that the direction of translational regulation is encoded in the 3′-UTR, not in the 5′-UTR. While much is known about 5′-UTR-dependent translational control in bacteria, the reported findings provide the first examples that both 5′- and 3′-UTRs are essential and sufficient to drive differential translational regulation in a prokaryote and therefore have to functionally interact in vivo. The current results indicate that 3′-UTR-dependent translational control had already evolved before capping and polyadenylation of transcripts were invented, which are essential for circularization of transcripts in eukaryotes.

Regulation of gvp genes encoding gas vesicle proteins in halophilic Archaea

Three gas vesicle gene clusters derived from Halobacterium salinarum (p-vac and c-vac) and Haloferax mediterranei (mc-vac) are used as model systems to study gene regulation in Archaea. An unusual pair of regulatory proteins is involved here, with GvpE acting as transcription activator and GvpD exhibiting a repressing function. Both regulators are able to interact leading to the loss of GvpE and the repression (or turnoff) of the gas vesicle formation. The latter function of GvpD requires a p-loop motif and an arginine-rich region, bR1. Both regulator proteins are differentially expressed from the same gvp transcript in Hfx. mediterranei and Hbt. salinarum PHH4. GvpE appears to recognize a 20-nucleotide activator sequence (UAS) located upstream and adjacent to the TFB-recognition element BRE of the two promoters driving the transcription of the divergently oriented gvpACNO and gvpDEFGHIJKLM gene clusters. The BRE elements of these two promoters are separated by 35 nucleotides only, and the distal portions of the two GvpE-UAS overlap considerably in the center of this region. Mutations here negatively affect the GvpE-induced activities of both gvp promoters, whereas alterations in the proximal UAS portions only affect the activity of the promoter located close by.

The molecular basis of salt adaptation in Methanosarcina mazei Gö1

The study on the molecular basis of salt adaptation and its regulation in archaea is still in its infancy, but genomics and functional genome analyses combined with classical biochemistry shed light on the processes conferring salt adaptation in the methanogenic archaeon Methanosarcina mazei Gö1. In this article, we will review discoveries made within the last years that will culminate in the description of the overall cellular response of M. mazei Gö1 to elevated salinities. This response includes accumulation of solutes and export of Na+ as well as potential uptake/export of K+ but also a restructuring of the cell surface.

Crystallization and preliminary X-ray diffraction analysis of the C-terminal domain of an archaeal transcription regulator

Little information is available on transcription regulation in Archaea, the third domain of life. Characterized archaeal regulators mostly belong to the Bacterial/Archaeal superfamily of Lrp-like transcription regulators (Lrp= Leucine-responsiveRegulatory Protein) [1]. One such example is Ss-LrpB from Sulfolobus solfataricus, which is a hyperthermoacidophilic organism growing optimally at 80°C and a pH between 2 and 3. Crystal structures of two Lrp-like proteins, both archaeal, have been determined: LrpA from Pyrococcus furiosus [2] and FL11 from PyrococcusOT3 [3]. Lrp-like proteins are composed of an N-terminal DNA-binding domain with a helix-turn-helix motif and a C-terminal ab-sandwich (babbab-fold), also called RAM domain (RAM=Regulation of Amino acid Metabolism). These two domains are connected by a flexible linker. Ss-LrpB binds its own control region [4], as do most Lrp-like proteins. This binding occurs cooperatively at three regularly spaced 15 bp binding sites with as consensus the palindromic sequence 5’-TTGYAWWWWWTRCAA-3’ (Y=pyrimidine, R=purine, W=weak bp). Due to the low solubility of Ss-LrpB, the C-terminal domain (comprising residues 69 to 147 of the full-length protein, omitting the hydrophobic linker) was cloned separately as a His-tag derivative and purified, aiming at a structural analysis. We succeeded in crystallizing this protein using the hanging-drop vapour-diffusionmethod in two different conditions [5]. Crystals, formed in one condition (100 mM NaPO4, 100 mM KPO4, 100 mM MES pH 6.5, 2.0 M NaCl), belong to the space group P21212 with unit-cell parameters a = 59.35 A; b = 74.86 A; c = 38.08 A and diffracted to 1.8 A resolution. Preliminary attempts at molecular replacement with the C-terminal part of LrpA as model (27.8 % amino acid identity, 55.7 % similarity), as well the dimer as the monomer, have not been successful. In order to solve the structure a Se-Met derivative is being prepared.

Phosphoenolpyruvate synthetase and pyruvate, phosphate dikinase of Thermoproteus tenax: key pieces in the puzzle of archaeal carbohydrate metabolism

The interconversion of phosphoenolpyruvate and pyruvate represents an important control point of the Embden-Meyerhof-Parnas (EMP) pathway in Bacteria and Eucarya, but little is known about this site of regulation in Archaea. Here we report on the coexistence of phosphoenolpyruvate synthetase (PEPS) and the first described archaeal pyruvate, phosphate dikinase (PPDK), which, besides pyruvate kinase (PK), are involved in the catalysis of this reaction in the hyperthermophilic crenarchaeote Thermoproteus tenax. The genes encoding T. tenax PEPS and PPDK were cloned and expressed in Escherichia coli, and the enzymic and regulatory properties of the recombinant gene products were analysed. Whereas PEPS catalyses the unidirectional conversion of pyruvate to phosphoenolpyruvate, PPDK shows a bidirectional activity with a preference for the catabolic reaction. In contrast to PK of T. tenax, which is regulated on transcript level but exhibits only limited regulatory potential on protein level, PEPS and PPDK activities are modulated by adenosine phosphates and intermediates of the carbohydrate metabolism. Additionally, expression of PEPS is regulated on transcript level in response to the offered carbon source as revealed by Northern blot analyses. The combined action of the differently regulated enzymes PEPS, PPDK and PK represents a novel way of controlling the interconversion of phosphoenolpyruvate and pyruvate in the reversible EMP pathway, allowing short-term and long-term adaptation to different trophic conditions. Comparative genomic analyses indicate the coexistence of PEPS, PPDK and PK in other Archaea as well, suggesting a similar regulation of the carbohydrate metabolism in these organisms.

Mechanism of Autoregulation by an Archaeal Transcriptional Repressor

The basal transcription machinery of archaea corresponds to the core components of the eucaryal RNA polymerase II apparatus. Thus, archaea possess a complex multi-subunit RNA polymerase, a TATA box-binding protein and a protein termed transcription factor B (TFB), which is a homologue of eucaryal transcription factor IIB (TFIIB). Intriguingly, archaeal genome sequencing projects have revealed the existence of homologues of bacterial transcriptional regulators. To investigate the mechanism of transcriptional regulation in archaea we have studied one such molecule, Lrs14, a Sulfolobus solfataricus P2 homologue of the bacterial leucine-responsive regulatory protein, Lrp. We find that purified Lrs14 specifically represses the transcription of its own gene in a reconstituted in vitro transcription system. Furthermore, we show that Lrs14 binding sites overlap the basal promoter elements of the Lrs14 promoter and reveal that binding of Lrs14 to these sites prevents promoter recognition by TATA box-binding protein and TFB.

Low temperature regulated DEAD-box RNA helicase from the antarctic archaeon, Methanococcoides burtonii

DEAD-box RNA helicases, by unwinding duplex RNA in bacteria and eukaryotes, are involved in essential cellular processes, including translation initiation and ribosome biogenesis, and have recently been implicated in enabling bacteria to survive cold-shock and grow at low temperature. Despite these critical physiological roles, they have not been characterized in archaea. Due to their presumed importance in removing cold-stabilised secondary structures in mRNA, we have characterised a putative DEAD-box RNA helicase gene ( deaD) from the Antarctic methanogen, Methanococcoides burtonii. The encoded protein, DeaD is predicted to contain a core element involved in ATP hydrolysis and RNA-binding, and an unusual C-terminal domain that contains seven perfect, trideca-peptide, direct repeats that may be involved in RNA binding. Alignment and phylogenetic analyses were performed on the core regions of the M. burtonii and other DEAD-box RNA helicases. These revealed a loose but consistent clustering of archaeal and bacterial sequences and enabled the generation of a prokaryotic-specific consensus sequence. The consensus highlights the importance of residues other than the eight motifs that are often associated with DEAD-box RNA helicases, as well as de-emphasising the importance of the “A” residue within the “DEAD” motif. Cells growing at 4°C contained abundant levels of deaD mRNA, however no mRNA was detected in cells growing at 23°C (the optimal temperature for growth). The transcription initiation site was mapped downstream from an archaeal box-A element (TATA box), which preceded a long (113 nucleotides) 5′-untranslated region (5′-UTR). Within the 5′-UTR was an 11 bp sequence that closely matches (nine out of 11) cold-box elements that are present in the 5′-UTRs of cold-shock induced genes from bacteria. To determine if the archaeal 5′-UTR performs an analagous function to the bacterial 5′-UTRs, the archaeal deaD 5′-UTR was transcribed in E. coli under the control of the cspA promoter and transcriptional terminator. It has previously been reported that overexpression of the cspA 5′-UTR leads to an extended cold-shock response due to the 5′-UTR titrating cellular levels of a cold-shock repressor protein(s). In our hands, the cold-shock protein profiles resulting from overexpression of Escherichia coli cspA and M. burtonii deaD 5′-UTRs were similar, however they did not differ from those for the overexpression of a control plasmid lacking a 5′-UTR. In association with other recent data from E. coli, our results indicate that the role of the 5′-UTR in gene regulation is presently unclear. Irrespective of the mechanisms, it is striking that highly similar 5′-UTRs with cold-box elements are present in cold induced genes from E. coli, Anabaena and M. burtonii. This is the first study examining low temperature regulation in archaea and provides initial evidence that gene expression from a cold adapted archaeon involves a bacterial-like transcriptional regulatory mechanism. In addition, it provides the foundation for further studies into the function and regulation of DEAD-box RNA helicases in archaea, and in particular, their roles in low temperature adaptation.

Transcriptional regulation in Archaea

Information regarding transcriptional regulation in Archaea has begun to emerge from in vivo genetic studies. Evidence to date suggests a varied repertoire of regulatory mechanisms in Archaea that invokes both bacterial and eukaryal paradigms, as well as some novel features. Overall simplicity of mechanisms may reflect the prokaryotic lifestyle. Sequencing projects suggest the existence of certain classes of regulators, but experimental verification is needed.

The alcohol dehydrogenase gene: distribution among Sulfolobales and regulation in Sulfolobus solfataricus

The distribution of the alcohol dehydrogenase gene ( adh) among different Archaea was investigated by Southern blot analysis revealing the potentiality of the adh gene as a specific marker for the genus Sulfolobus. Moreover, the in vivo expression of the adh gene from a new isolate of Sulfolobus solfataricus, Gθ, was studied to investigate gene regulation in Archaea. Primer extension analysis allowed the identification of a single initiation site and the TATA box element. Comparison of the Gθ adh promoter with the corresponding Ssadh ( adh from S. solfataricus) and RC3adh ( adh from Sulfolobus RC3) also revealed the presence of two putative regulatory inverted repeats at the 5′ of the TATA element. Northern blot analysis and enzymatic assays demonstrated that the transcription and expression of the Gθ adh gene is regulated by different carbon and energy sources or by the natural substrate of the ADH enzyme.

The alcohol dehydrogenase gene: distribution among Sulfolobales and regulation in Sulfolobus solfataricus.

The distribution of the alcohol dehydrogenase gene (adh) among different Archaea was investigated by Southern blot analysis revealing the potentiality of the adh gene as a specific marker for the genus Sulfolobus. Moreover, the in vivo expression of the adh gene from a new isolate of Sulfolobus solfataricus, G theta, was studied to investigate gene regulation in Archaea. Primer extension analysis allowed the identification of a single initiation site and the TATA box element. Comparison of the G theta adh promoter with the corresponding Ssadh (adh from S. solfataricus) and RC3adh (adh from Sulfolobus RC3) also revealed the presence of two putative regulatory inverted repeats at the 5' of the TATA element. Northern blot analysis and enzymatic assays demonstrated that the transcription and expression of the G theta adh gene is regulated by different carbon and energy sources or by the natural substrate of the ADH enzyme.