Distinct Regions of the Haloferax volcanii Dolichol Phosphate-Mannose Synthase AglD Mediate the Assembly and Subsequent Processing of the Lipid-Linked Mannose.

Haloferax volcanii AglD is currently the only archaeal dolichol phosphate (DolP)-mannose synthase shown to participate in N-glycosylation. However, the relation between AglD and Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase for which structural information is presently available, was unclear. In this report, similarities between the PF0058 and AglD catalytic domains were revealed. At the same time, AglD includes a transmembrane domain far longer than that of PF0058 or other DolP-mannose synthases. To determine whether this extension affords AglD functions in addition to generating mannose-charged DolP, a series of Hfx. volcanii strains expressing truncated versions of AglD was generated. Mass spectrometry revealed that a version of AglD comprising the catalytic domain and only two of the six to nine predicted membrane-spanning domains could mediate mannose addition to DolP. However, in cells expressing this or other truncated versions of AglD, mannose was not transferred from the lipid to the protein-bound tetrasaccharide precursor of the N-linked pentasaccharide normally decorating Hfx. volcanii glycoproteins. These results thus point to AglD as contributing to additional aspects of Hfx. volcanii N-glycosylation beyond charging DolP with mannose. Accordingly, the possibility that AglD, possibly in coordination with AglR, translocates DolP-mannose across the plasma membrane is discussed.

Expression and tandem affinity purification of 20S proteasomes and other multisubunit complexes in Haloferax volcanii.

Tandem affinity purification is a useful strategy to isolate multisubunit complexes of high yield and purity but can be limited when working with halophilic proteins that are not properly expressed in Escherichia coli. Halophilic proteins are desirable for bioindustrial applications as they are often stable and active in organic solvents; however, these proteins can be difficult to express, fold, and purify by traditional technologies. Haloarchaea provide a useful alternative for expression of halophilic proteins. These microorganisms use a salt-in strategy to maintain homeostasis and express most of their proteins with halophilic properties and low pI. Here, we provide detailed protocols for the genetic modification, expression and tandem affinity purification of "salt-loving" multisubunit complexes from the haloarchaeon Haloferax volcanii. The strategy for isolation of affinity tagged 20S proteasomes that form cylindrical proteolytic nanomachines of α1, α2 and ß subunits is described.

Glucose Metabolism and Acetate Switch in Archaea: the Enzymes in Haloferax volcanii.

The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. Following our previous studies on key enzymes of this pathway, we now focus on the characterization of enzymes involved in 3-phosphoglycerate conversion to pyruvate, in anaplerosis, and in acetyl coenzyme A (acetyl-CoA) formation from pyruvate. These enzymes include phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenolpyruvate carboxylase, and pyruvate-ferredoxin oxidoreductase. The essential function of these enzymes were shown by transcript analyses and growth experiments with respective deletion mutants. Furthermore, we show that H. volcanii-during aerobic growth on glucose-excreted significant amounts of acetate, which was consumed in the stationary phase (acetate switch). The enzyme catalyzing the conversion of acetyl-CoA to acetate as part of the acetate overflow mechanism, an ADP-forming acetyl-CoA synthetase (ACD), was characterized. The functional involvement of ACD in acetate formation and of AMP-forming acetyl-CoA synthetases (ACSs) in activation of excreted acetate was proven by using respective deletion mutants. Together, the data provide a comprehensive analysis of enzymes of the spED pathway and of anaplerosis and report the first genetic evidence of the functional involvement of enzymes of the acetate switch in archaea.IMPORTANCE In this work, we provide a comprehensive analysis of glucose degradation via the semiphosphorylative Entner-Doudoroff pathway in the haloarchaeal model organism Haloferax volcanii The study includes transcriptional analyses, growth experiments with deletion mutants. and characterization of all enzymes involved in the conversion of 3-phosphoglycerate to acetyl coenzyme A (acetyl-CoA) and in anaplerosis. Phylogenetic analyses of several enzymes indicate various lateral gene transfer events from bacteria to haloarchaea. Furthermore, we analyzed the key players involved in the acetate switch, i.e., in the formation (overflow) and subsequent consumption of acetate during aerobic growth on glucose. Together, the data provide novel aspects of glucose degradation, anaplerosis, and acetate switch in H. volcanii and thus expand our understanding of the unusual sugar metabolism in archaea.

Insights into synthesis and function of KsgA/Dim1-dependent rRNA modifications in archaea.

Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.

Protein Splicing Activity of the Haloferax volcanii PolB-c Intein Is Sensitive to Homing Endonuclease Domain Mutations.

Inteins are selfish genetic elements residing in open reading frames that can splice post-translationally, resulting in the ligation of an uninterrupted, functional protein. Like other inteins, the DNA polymerase B (PolB) intein of the halophilic archaeon Haloferax volcanii has an active homing endonuclease (HEN) domain, facilitating its horizontal transmission. Previous work has shown that the presence of the PolB intein exerts a significant fitness cost on the organism compared to an intein-free isogenic H. volcanii. Here, we show that mutation of a conserved residue in the HEN domain not only reduces intein homing but also slows growth. Surprisingly, although this mutation is far from the protein splicing active site, it also significantly reduces in vitro protein splicing. Moreover, two additional HEN domain mutations, which could not be introduced to H. volcanii, presumably due to lethality, also eliminate protein splicing activity in vitro. These results suggest an interplay between HEN residues and the protein splicing domain, despite an over 35 Å separation in a PolB intein homology model. The combination of in vivo and in vitro evidence strongly supports a model of codependence between the self-splicing domain and the HEN domain that has been alluded to by previous in vitro studies of protein splicing with HEN domain-containing inteins.

Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea.

The archaeal cytoplasmic membrane provides an anchor for many surface proteins. Recently, a novel membrane anchoring mechanism involving a peptidase, archaeosortase A (ArtA), and C-terminal lipid attachment of surface proteins was identified in the model archaeon Haloferax volcanii ArtA is required for optimal cell growth and morphogenesis, and the S-layer glycoprotein (SLG), the sole component of the H. volcanii cell wall, is one of the targets for this anchoring mechanism. However, how exactly ArtA function and regulation control cell growth and morphogenesis is still elusive. Here, we report that archaeal homologs to the bacterial phosphatidylserine synthase (PssA) and phosphatidylserine decarboxylase (PssD) are involved in ArtA-dependent protein maturation. Haloferax volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth, and morphological phenotypes similar to those of an ΔartA mutant. Moreover, we showed a loss of covalent lipid attachment to SLG in the ΔhvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the ΔhvpssA and ΔhvpssD mutant strains. Strikingly, ArtA, HvPssA, and HvPssD green fluorescent protein (GFP) fusions colocalized to the midcell position of H. volcanii cells, strongly supporting that they are involved in the same pathway. Finally, we have shown that the SLG is also recruited to the midcell before being secreted and lipid anchored at the cell outer surface. Collectively, our data suggest that haloarchaea use the midcell as the main surface processing hot spot for cell elongation, division, and shape determination.IMPORTANCE The subcellular organization of biochemical processes in space and time is still one of the most mysterious topics in archaeal cell biology. Despite the fact that haloarchaea largely rely on covalent lipid anchoring to coat the cell envelope, little is known about how cells coordinate de novo synthesis and about the insertion of this proteinaceous layer throughout the cell cycle. Here, we report the identification of two novel contributors to ArtA-dependent lipid-mediated protein anchoring to the cell surface, HvPssA and HvPssD. ArtA, HvPssA, and HvPssD, as well as SLG, showed midcell localization during growth and cytokinesis, indicating that haloarchaeal cells confine phospholipid processing in order to promote midcell elongation. Our findings have important implications for the biogenesis of the cell surface.

D-galactose catabolism in archaea: operation of the DeLey-Doudoroff pathway in Haloferax volcanii.

The haloarchaeon Haloferax volcanii was found to grow on D-galactose as carbon and energy source. Here we report a comprehensive analysis of D-galactose catabolism in H. volcanii. Genome analyses indicated a cluster of genes encoding putative enzymes of the DeLey-Doudoroff pathway for D-galactose degradation including galactose dehydrogenase, galactonate dehydratase, 2-keto-3-deoxygalactonate kinase and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase. The recombinant galactose dehydrogenase and galactonate dehydratase showed high specificity for D-galactose and galactonate, respectively, whereas KDPGal aldolase was promiscuous in utilizing KDPGal and also the C4 epimer 2-keto-3-deoxy-6-phosphogluconate as substrates. Growth studies with knock-out mutants indicated the functional involvement of galactose dehydrogenase, galactonate dehydratase and KDPGal aldolase in D-galactose degradation. Further, the transcriptional regulator GacR was identified, which was characterized as an activator of genes of the DeLey-Doudoroff pathway. Finally, genes were identified encoding components of an ABC transporter and a knock-out mutant of the substrate binding protein indicated the functional involvement of this transporter in D-galactose uptake. This is the first report of D-galactose degradation via the DeLey-Doudoroff pathway in the domain of archaea.

Haloferax volcanii for biotechnology applications: challenges, current state and perspectives.

Haloferax volcanii is an obligate halophilic archaeon with its origin in the Dead Sea. Simple laboratory culture conditions and a wide range of genetic tools have made it a model organism for studying haloarchaeal cell biology. Halophilic enzymes of potential interest to biotechnology have opened up the application of this organism in biocatalysis, bioremediation, nanobiotechnology, bioplastics and the biofuel industry. Functionally active halophilic proteins can be easily expressed in a halophilic environment, and an extensive genetic toolkit with options for regulated protein overexpression has allowed the purification of biotechnologically important enzymes from different halophiles in H. volcanii. However, corrosion mediated damage caused to stainless-steel bioreactors by high salt concentrations and a tendency to form biofilms when cultured in high volume are some of the challenges of applying H. volcanii in biotechnology. The ability to employ expressed active proteins in immobilized cells within a porous biocompatible matrix offers new avenues for exploiting H. volcanii in biotechnology. This review critically evaluates the various application potentials, challenges and toolkits available for using this extreme halophilic organism in biotechnology.

Homologs of aquifex aeolicus protein-only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei.

The mature 5'-ends of tRNAs are generated by RNase P in all domains of life. The ancient form of the enzyme is a ribonucleoprotein consisting of a catalytic RNA and one or more protein subunits. However, in the hyperthermophilic bacterium Aquifex aeolicus and close relatives, RNase P is a protein-only enzyme consisting of a single type of polypeptide (Aq_880, ~23 kDa). In many archaea, homologs of Aq_880 were identified (termed HARPs for Homologs of Aquifex RNase P) in addition to the RNA-based RNase P, raising the question about the functions of HARP and the classical RNase P in these archaea. Here we investigated HARPs from two euryarchaeotes, Haloferax volcanii and Methanosarcina mazei. Archaeal strains with HARP gene knockouts showed no growth phenotypes under standard conditions, temperature and salt stress (H. volcanii) or nitrogen deficiency (M. mazei). Recombinant H. volcanii and M. mazei HARPs were basically able to catalyse specific tRNA 5'-end maturation in vitro. Furthermore, M. mazei HARP was able to rescue growth of an Escherichia coli RNase P depletion strain with comparable efficiency as Aq_880, while H. volcanii HARP was unable to do so. In conclusion, both archaeal HARPs showed the capacity (in at least one functional assay) to act as RNases P. However, the ease to obtain knockouts of the singular HARP genes and the lack of growth phenotypes upon HARP gene deletion contrasts with the findings that the canonical RNase P RNA gene cannot be deleted in H. volcanii, and a knockdown of RNase P RNA in H. volcanii results in severe tRNA processing defects. We conclude that archaeal HARPs do not make a major contribution to global tRNA 5'-end maturation in archaea, but may well exert a specialised, yet unknown function in (t)RNA metabolism. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1109-1116, 2019.

Rhodanese-Like Domain Protein UbaC and Its Role in Ubiquitin-Like Protein Modification and Sulfur Mobilization in Archaea.

Ubiquitin-like protein (Ubl) modification targets proteins for transient inactivation and/or proteasome-mediated degradation in archaea. Here the rhodanese-like domain (RHD) protein UbaC (HVO_1947) was found to copurify with the E1-like enzyme (UbaA) of the Ubl modification machinery in the archaeon Haloferax volcanii UbaC was shown to be important for Ubl ligation, particularly for the attachment of the Ubl SAMP2/3s to protein targets after exposure to oxidants (NaOCl, dimethyl sulfoxide [DMSO], and methionine sulfoxide [MetO]) and the proteasome inhibitor bortezomib. While UbaC was needed for ligation of the Ubl SAMP1 to MoaE (the large subunit of molybdopterin synthase), it was not important in the formation of oxidant-induced SAMP1 protein conjugates. Indicative of defects in sulfur relay, mutation of ubaC impaired molybdenum cofactor (Moco)-dependent DMSO reductase activity and cell survival at elevated temperature, suggesting a correlation with defects in the 2-thiolated state of wobble uridine tRNA. Overall, the archaeal stand-alone RHD UbaC has an important function in Ubl ligation and is associated with sulfur relay processes.IMPORTANCE Canonical E2 Ub/Ubl-conjugating enzymes are not conserved in the dual-function Ubl systems associated with protein modification and sulfur relay. Instead, the C-terminal RHDs of E1-RHD fusion proteins are the apparent E2 modules of these systems in eukaryotes. E1s that lack an RHD are common in archaea. Here we identified an RHD (UbaC) that serves as an apparent E2 analog with the E1-like UbaA in the dual-function Ubl sampylation system of archaea. Unlike the eukaryotic E1-RHD fusion, the archaeal RHD is a stand-alone protein. This new insight suggests that E1 function in Ubl pathways could be influenced by shifts in RHD abundance and/or competition with other protein partners in the cell.

Characterisation of a solvent-tolerant haloarchaeal (R)-selective transaminase isolated from a Triassic period salt mine.

Transaminase enzymes (TAms) are becoming increasingly valuable in the chemist's toolbox as a biocatalytic route to chiral amines. Despite high profile successes, the lack of (R)-selective TAms and robustness under harsh industrial conditions continue to prove problematic. Herein, we report the isolation of the first haloarchaeal TAm (BC61-TAm) to be characterised for the purposes of pharmaceutical biocatalysis. BC61-TAm is an (R)-selective enzyme, cloned from an extremely halophilic archaeon, isolated from a Triassic period salt mine. Produced using a Haloferax volcanii-based expression model, the resulting protein displays a classic halophilic activity profile, as well as thermotolerance (optimum 50 °C) and organic solvent tolerance. Molecular modelling predicts the putative active site residues of haloarchaeal TAms, with molecular dynamics simulations providing insights on the basis of BC61-TAm's organic solvent tolerance. These results represent an exciting advance in the study of transaminases from extremophiles, providing a possible scaffold for future discovery of biocatalytic enzymes with robust properties.

Cyclic nucleotides in archaea: Cyclic di-AMP in the archaeon Haloferax volcanii and its putative role.

The role of cyclic nucleotides as second messengers for intracellular signal transduction has been well described in bacteria. One recently discovered bacterial second messenger is cyclic di-adenylate monophosphate (c-di-AMP), which has been demonstrated to be essential in bacteria. Compared to bacteria, significantly less is known about second messengers in archaea. This study presents the first evidence of in vivo presence of c-di-AMP in an archaeon. The model organism Haloferax volcanii was demonstrated to produce c-di-AMP. Its genome encodes one diadenylate cyclase (DacZ) which was shown to produce c-di-AMP in vitro. Similar to bacteria, the dacZ gene is essential and homologous overexpression of DacZ leads to cell death, suggesting the need for tight regulation of c-di-AMP levels. Such tight regulation often indicates the control of important regulatory processes. A central target of c-di-AMP signaling in bacteria is cellular osmohomeostasis. The results presented here suggest a comparable function in H. volcanii. A strain with decreased c-di-AMP levels exhibited an increased cell area in hypo-salt medium, implying impaired osmoregulation. In summary, this study expands the field of research on c-di-AMP and its physiological function to archaea and indicates that osmoregulation is likely to be a common function of c-di-AMP in bacteria and archaea.

Haloferax volcanii as immobilised whole cell biocatalyst: new applications for halophilic systems.

Enzyme-mediated synthesis of pharmaceutical compounds is a 'green' alternative to traditional synthetic chemistry, and microbial engineering opens up the possibility of using whole cells as mini-factories. Whole-cell biocatalysis reduces cost by eliminating expensive enzyme purification and cofactor addition steps, as well as resulting in increased enzyme stability. Haloferax volcanii is a model halophilic archaeon encoding highly salt and organic solvent tolerant enzymes such as alcohol dehydrogenase (HvADH2), which catalyses the reduction of aldehydes and ketone in the presence of NADPH/NADH cofactor. A H. volcanii strain for constitutive HvADH2 expression was generated using a strong synthetic promoter (p.syn). The strain was immobilised in calcium alginate beads and repeatedly used as a whole-cell biocatalyst. The reduction of acetophenone, used as test substrate, was very successful and high yields were detected from immobilised whole cells over repeated biotransformation cycles. The immobilised H. volcanii retained stability and high product yields after 1 month of storage at room temperature. This newly developed system offers halophilic enzyme expression in its native environment, high product yield, stability and reusability without the addition of any expensive NADPH/NADH cofactor. This is the first report of whole cell-mediated biocatalysis by the halophilic archaeon H. volcanii.

l-Rhamnose catabolism in archaea.

The halophilic archaeon Haloferax volcanii utilizes l-rhamnose as a sole carbon and energy source. It is shown that l-rhamnose is taken up by an ABC transporter and is oxidatively degraded to pyruvate and l-lactate via the diketo-hydrolase pathway. The genes involved in l-rhamnose uptake and degradation form a l-rhamnose catabolism (rhc) gene cluster. The rhc cluster also contains a gene, rhcR, that encodes the transcriptional regulator RhcR which was characterized as an activator of all rhc genes. 2-keto-3-deoxy-l-rhamnonate, a metabolic intermediate of l-rhamnose degradation, was identified as inducer molecule of RhcR. The essential function of rhc genes for uptake and degradation of l-rhamnose was proven by the respective knockout mutants. Enzymes of the diketo-hydrolase pathway, including l-rhamnose dehydrogenase, l-rhamnonolactonase, l-rhamnonate dehydratase, 2-keto-3-deoxy-l-rhamnonate dehydrogenase and 2,4-diketo-3-deoxy-l-rhamnonate hydrolase, were characterized. Further, genes of the diketo-hydrolase pathway were also identified in the hyperthermophilic crenarchaeota Vulcanisaeta distributa and Sulfolobus solfataricus and selected enzymes were characterized, indicating the presence of the diketo-hydrolase pathway in these archaea. Together, this is the first comprehensive description of l-rhamnose catabolism in the domain of archaea.

Catalytic linkage between caspase activity and proteostasis in Archaea.

The model haloarchaeon, Haloferax volcanii possess an extremely high, and highly specific, basal caspase activity in exponentially growing cells that closely resembles caspase-4. This activity is specifically inhibited by the pan-caspase inhibitor, z-VAD-FMK, and has no cross-reactivity with other known protease families. Although it is one of the dominant cellular proteolytic activities in exponentially growing H. volcanii cells, the interactive cellular roles remain unknown and the protein(s) responsible for this activity remain elusive. Here, biochemical purification and in situ trapping with caspase targeted covalent inhibitors combined with genome-enabled proteomics, structural analysis, targeted gene knockouts and treatment with canavanine demonstrated a catalytic linkage between caspase activity and thermosomes, proteasomes and cdc48b, a cell division protein and proteasomal degradation facilitating ATPase, as part of an 'interactase' of stress-related protein complexes with an established link to the unfolded protein response (UPR). Our findings provide novel cellular and biochemical context for the observed caspase activity in Archaea and add new insight to understanding the role of this activity, implicating their possible role in the establishment of protein stress and ER associated degradation pathways in Eukarya.

The trimeric Hef-associated nuclease HAN is a 3'→5' exonuclease and is probably involved in DNA repair.

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN's function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3'-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3'-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.

Evolutionary insights into Trm112-methyltransferase holoenzymes involved in translation between archaea and eukaryotes.

Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms.

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.

Two distinct glyceraldehyde-3-phosphate dehydrogenases in glycolysis and gluconeogenesis in the archaeon Haloferax volcanii.

The halophilic archaeon Haloferax volcanii degrades glucose via the semiphosphorylative Entner-Doudoroff pathway and can also grow on gluconeogenic substrates. Here, the enzymes catalysing the conversion of glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate were analysed. The genome contains the genes gapI and gapII encoding two putative GAP dehydrogenases, and pgk encoding phosphoglycerate kinase (PGK). We show that gapI is functionally involved in sugar catabolism, whereas gapII is involved in gluconeogenesis. For pgk, an amphibolic function is indicated. This is the first report of the functional involvement of a phosphorylating glyceraldehyde-3-phosphate dehydrogenase and PGK in sugar catabolism in archaea. Phylogenetic analyses indicate that the catabolic gapI from H. volcanii is acquired from bacteria via lateral genetransfer, whereas the anabolic gapII as well as pgk are of archaeal origin.

LonB Protease Is a Novel Regulator of Carotenogenesis Controlling Degradation of Phytoene Synthase in Haloferax volcanii.

The membrane protease LonB is an essential protein in the archaeon Haloferax volcanii and globally impacts its physiology. However, natural substrates of the archaeal Lon protease have not been identified. The whole proteome turnover was examined in a H. volcanii LonB mutant under reduced and physiological protease levels. LC-MS/MS combined with stable isotope labeling was applied for the identification/quantitation of membrane and cytoplasm proteins. Differential synthesis and degradation rates were evidenced for 414 proteins in response to Lon expression. A total of 58 proteins involved in diverse cellular processes showed a degradation pattern (none/very little degradation in the absence of Lon and increased degradation in the presence of Lon) consistent with a LonB substrate, which was further substantiated for several of these candidates by pull-down assays. The most notable was phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthesis. The rapid degradation of PSY upon LonB induction in addition to the remarkable stabilization of this protein and hyperpigmentation phenotype in the Lon mutant strongly suggest that PSY is a LonB substrate. This work identifies for the first time candidate targets of the archaeal Lon protease and establishes proteolysis by Lon as a novel post-translational regulatory mechanism of carotenogenesis.