Alanine Tails Signal Proteolysis in Bacterial Ribosome-Associated Quality Control.

In ribosome-associated quality control (RQC), Rqc2/NEMF closely supports the E3 ligase Ltn1/listerin in promoting ubiquitylation and degradation of aberrant nascent-chains obstructing large (60S) ribosomal subunits-products of ribosome stalling during translation. However, while Ltn1 is eukaryote-specific, Rqc2 homologs are also found in bacteria and archaea; whether prokaryotic Rqc2 has an RQC-related function has remained unknown. Here, we show that, as in eukaryotes, a bacterial Rqc2 homolog (RqcH) recognizes obstructed 50S subunits and promotes nascent-chain proteolysis. Unexpectedly, RqcH marks nascent-chains for degradation in a direct manner, by appending C-terminal poly-alanine tails that act as degrons recognized by the ClpXP protease. Furthermore, RqcH acts redundantly with tmRNA/ssrA and protects cells against translational and environmental stresses. Our results uncover a proteolytic-tagging mechanism with implications toward the function of related modifications in eukaryotes and suggest that RQC was already active in the last universal common ancestor (LUCA) to help cope with incomplete translation.

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

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

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

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

Discovery of a novel transcriptional regulator of sugar catabolism in archaea.

The haloarchaeon Haloferax volcanii degrades D-glucose via the semiphosphorylative Entner-Doudoroff pathway and D-fructose via a modified Embden-Meyerhof pathway. Here, we report the identification of GfcR, a novel type of transcriptional regulator that functions as an activator of both D-glucose and D-fructose catabolism. We find that in the presence of D-glucose, GfcR activates gluconate dehydratase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase and also acts as activator of the phosphotransferase system and of fructose-1,6-bisphosphate aldolase, which are involved in uptake and degradation of D-fructose. In addition, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase are activated by GfcR in the presence of D-fructose and also during growth on D-galactose and glycerol. Electrophoretic mobility shift assays indicate that GfcR binds directly to promoters of regulated genes. Specific intermediates of the degradation pathways of the three hexoses and of glycerol were identified as inducer molecules of GfcR. GfcR is composed of a phosphoribosyltransferase (PRT) domain with an N-terminal helix-turn-helix motif and thus shows homology to PurR of Gram-positive bacteria that is involved in the transcriptional regulation of nucleotide biosynthesis. We propose that GfcR of H. volcanii evolved from a PRT-like enzyme to attain a function as a transcriptional regulator of central sugar catabolic pathways in archaea.

The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNAPyl pairs in the same organism.

Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNAPyl pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNAPyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNAPyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNAPyl2 charging by PylRS2 is defined by the enzyme's shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNAPyl pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure.

Halophilic archaea and their potential to generate renewable fuels and chemicals.

Lignocellulosic biofuels and chemicals have great potential to reduce our dependence on fossil fuels and mitigate air pollution by cutting down on greenhouse gas emissions. Chemical, thermal, and enzymatic processes are used to release the sugars from the lignocellulosic biomass for conversion to biofuels. These processes often operate at extreme pH conditions, high salt concentrations, and/or high temperature. These harsh treatments add to the cost of the biofuels, as most known biocatalysts do not operate under these conditions. To increase the economic feasibility of biofuel production, microorganisms that thrive in extreme conditions are considered as ideal resources to generate biofuels and value-added products. Halophilic archaea (haloarchaea) are isolated from hypersaline ecosystems with high salt concentrations approaching saturation (1.5-5 M salt concentration) including environments with extremes in pH and/or temperature. The unique traits of haloarchaea and their enzymes that enable them to sustain catalytic activity in these environments make them attractive resources for use in bioconversion processes that must occur across a wide range of industrial conditions. Biocatalysts (enzymes) derived from haloarchaea occupy a unique niche in organic solvent, salt-based, and detergent industries. This review focuses on the use of haloarchaea and their enzymes to develop and improve biofuel production. The review also highlights how haloarchaea produce value-added products, such as antibiotics, carotenoids, and bioplastic precursors, and can do so using feedstocks considered "too salty" for most microbial processes including wastes from the olive-mill, shell fish, and biodiesel industries.

Putting phage to work in deubiquitinase ligand discovery.

Inhibiting deubiquitinase (DUB) function is a promising strategy for the treatment of cancers and other human diseases. Of the hundreds of human DUBs, USP11 has emerged as an ideal therapeutic target, as it regulates DNA double-strand break repair by homologous recombination (HR) and other functions central to eukaryotic cell survival. A new study by Spiliotopoulos et al. cleverly uses next-generation phage display (NGPD) to identify peptide ligands that bind USP11 in a unique pocket that impacts HR. The study provides an important step toward novel DUB inhibitors that may reduce the resistance of some cancers to current treatment options.

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.

Prokaryotic ubiquitin-like protein modification.

Prokaryotes form ubiquitin (Ub)-like isopeptide bonds on the lysine residues of proteins by at least two distinct pathways that are reversible and regulated. In mycobacteria, the C-terminal Gln of Pup (prokaryotic ubiquitin-like protein) is deamidated and isopeptide linked to proteins by a mechanism distinct from ubiquitylation in enzymology yet analogous to ubiquitylation in targeting proteins for destruction by proteasomes. Ub-fold proteins of archaea (SAMPs, small archaeal modifier proteins) and Thermus (TtuB, tRNA-two-thiouridine B) that differ from Ub in amino acid sequence, yet share a common ß-grasp fold, also form isopeptide bonds by a mechanism that appears streamlined compared with ubiquitylation. SAMPs and TtuB are found to be members of a small group of Ub-fold proteins that function not only in protein modification but also in sulfur-transfer pathways associated with tRNA thiolation and molybdopterin biosynthesis. These multifunctional Ub-fold proteins are thought to be some of the most ancient of Ub-like protein modifiers.

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

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

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

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

Multiplex quantitative SILAC for analysis of archaeal proteomes: a case study of oxidative stress responses.

Stable isotope labelling of amino acids in cell culture (SILAC) is a quantitative proteomic method that can illuminate new pathways used by cells to adapt to different lifestyles and niches. Archaea, while thriving in extreme environments and accounting for ∼20%-40% of the Earth's biomass, have not been analyzed with the full potential of SILAC. Here, we report SILAC for quantitative comparison of archaeal proteomes, using Haloferax volcanii as a model. A double auxotroph was generated that allowed for complete incorporation of 13 C/15 N-lysine and 13 C-arginine such that each peptide derived from trypsin digestion was labelled. This strain was found amenable to multiplex SILAC by case study of responses to oxidative stress by hypochlorite. A total of 2565 proteins was identified by LC-MS/MS analysis (q-value ≤ 0.01) that accounted for 64% of the theoretical proteome. Of these, 176 proteins were altered at least 1.5-fold (p-value < 0.05) in abundance during hypochlorite stress. Many of the differential proteins were of unknown function. Those of known function included transcription factor homologs related to oxidative stress by 3D-homology modelling and orthologous group comparisons. Thus, SILAC is found to be an ideal method for quantitative proteomics of archaea that holds promise to unravel gene function.

A Cobalamin Activity-Based Probe Enables Microbial Cell Growth and Finds New Cobalamin-Protein Interactions across Domains.

Understanding the factors that regulate microbe function and microbial community assembly, function, and fitness is a grand challenge. A critical factor and an important enzyme cofactor and regulator of gene expression is cobalamin (vitamin B12). Our knowledge of the roles of vitamin B12 is limited, because technologies that enable in situ characterization of microbial metabolism and gene regulation with minimal impact on cell physiology are needed. To meet this need, we show that a synthetic probe mimic of B12 supports the growth of B12-auxotrophic bacteria and archaea. We demonstrate that a B12 activity-based probe (B12-ABP) is actively transported into Escherichia coli cells and converted to adenosyl-B12-ABP akin to native B12 Identification of the proteins that bind the B12-ABP in vivo in E. coli, a Rhodobacteraceae sp. and Haloferax volcanii, demonstrate the specificity for known and novel B12 protein targets. The B12-ABP also regulates the B12 dependent RNA riboswitch btuB and the transcription factor EutR. Our results demonstrate a new approach to gain knowledge about the role of B12 in microbe functions. Our approach provides a powerful nondisruptive tool to analyze B12 interactions in living cells and can be used to discover the role of B12 in diverse microbial systems.IMPORTANCE We demonstrate that a cobalamin chemical probe can be used to investigate in vivo roles of vitamin B12 in microbial growth and regulation by supporting the growth of B12 auxotrophic bacteria and archaea, enabling biological activity with three different cell macromolecules (RNA, DNA, and proteins), and facilitating functional proteomics to characterize B12-protein interactions. The B12-ABP is both transcriptionally and translationally able to regulate gene expression analogous to natural vitamin B12 The application of the B12-ABP at biologically relevant concentrations facilitates a unique way to measure B12 microbial dynamics and identify new B12 protein targets in bacteria and archaea. We demonstrate that the B12-ABP can be used to identify in vivo protein interactions across diverse microbes, from E. coli to microbes isolated from naturally occurring phototrophic biofilms to the salt-tolerant archaea Haloferax volcanii.

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.

Proteome targets of ubiquitin-like samp1ylation are associated with sulfur metabolism and oxidative stress in Haloferax volcanii.

Small archeal modifier proteins (SAMPs) are related to ubiquitin in tertiary structure and in their isopeptide linkage to substrate proteins. SAMPs also function in sulfur mobilization to form biomolecules such as molybdopterin and thiolated tRNA. While SAMP1 is essential for anaerobic growth and covalently attached to lysine residues of its molybdopterin synthase partner MoaE (K240 and K247), the full diversity of proteins modified by samp1ylation is not known. Here, we expand the knowledge of proteins isopeptide linked to SAMP1. LC-MS/MS analysis of -Gly-Gly signatures derived from SAMP1 S85R conjugates cleaved with trypsin was used to detect sites of sampylation (23 lysine residues) that mapped to 11 target proteins. Many of the identified target proteins were associated with sulfur metabolism and oxidative stress including MoaE, SAMP-activating E1 enzyme (UbaA), methionine sulfoxide reductase homologs (MsrA and MsrB), and the Fe-S assembly protein SufB. Several proteins were found to have multiple sites of samp1ylation, and the isopeptide linkage at SAMP3 lysines (K18, K55, and K62) revealed hetero-SAMP chain topologies. Follow-up affinity purification of selected protein targets (UbaA and MoaE) confirmed the LC-MS/MS results. 3D homology modeling suggested sampy1ylation is autoregulatory in inhibiting the activity of its protein partners (UbaA and MoaE), while occurring on the surface of some protein targets, such as SufB and MsrA/B. Overall, we provide evidence that SAMP1 is a ubiquitin-like protein modifier that is relatively specific in tagging its protein partners as well as proteins associated with oxidative stress response.

Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents.

Soluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PPi) to orthophosphate (Pi) are commonly used to accelerate and detect biosynthetic reactions that generate PPi as a by-product. Current PPAs are inactivated by high salt concentrations and organic solvents, which limits the extent of their use. Here we report a class A type PPA of the haloarchaeon Haloferax volcanii (HvPPA) that is thermostable and displays robust PPi-hydrolyzing activity under conditions of 25% (vol/vol) organic solvent and salt concentrations from 25 mM to 3 M. HvPPA was purified to homogeneity as a homohexamer by a rapid two-step method and was found to display non-Michaelis-Menten kinetics with a Vmax of 465 U · mg(-1) for PPi hydrolysis (optimal at 42°C and pH 8.5) and Hill coefficients that indicated cooperative binding to PPi and Mg(2+). Similarly to other class A type PPAs, HvPPA was inhibited by sodium fluoride; however, hierarchical clustering and three-dimensional (3D) homology modeling revealed HvPPA to be distinct in structure from characterized PPAs. In particular, HvPPA was highly negative in surface charge, which explained its extreme resistance to organic solvents. To demonstrate that HvPPA could drive thermodynamically unfavorable reactions to completion under conditions of reduced water activity, a novel coupled assay was developed; HvPPA hydrolyzed the PPi by-product generated in 2 M NaCl by UbaA (a "salt-loving" noncanonical E1 enzyme that adenylates ubiquitin-like proteins in the presence of ATP). Overall, we demonstrate HvPPA to be useful for hydrolyzing PPi under conditions of reduced water activity that are a hurdle to current PPA-based technologies.

Ubiquitin-like small archaeal modifier proteins (SAMPs) in Haloferax volcanii.

Archaea, one of three major evolutionary lineages of life, encode proteasomes highly related to those of eukaryotes. In contrast, archaeal ubiquitin-like proteins are less conserved and not known to function in protein conjugation. This has complicated our understanding of the origins of ubiquitination and its connection to proteasomes. Here we report two small archaeal modifier proteins, SAMP1 and SAMP2, with a beta-grasp fold and carboxy-terminal diglycine motif similar to ubiquitin, that form protein conjugates in the archaeon Haloferax volcanii. The levels of SAMP-conjugates were altered by nitrogen-limitation and proteasomal gene knockout and spanned various functions including components of the Urm1 pathway. LC-MS/MS-based collision-induced dissociation demonstrated isopeptide bonds between the C-terminal glycine of SAMP2 and the epsilon-amino group of lysines from a number of protein targets and Lys 58 of SAMP2 itself, revealing poly-SAMP chains. The widespread distribution and diversity of pathways modified by SAMPylation suggest that this type of protein conjugation is central to the archaeal lineage.

Archaeal ubiquitin-like SAMP3 is isopeptide-linked to proteins via a UbaA-dependent mechanism.

SAMP1 and SAMP2 are ubiquitin-like proteins that function as protein modifiers and are required for the production of sulfur-containing biomolecules in the archaeon Haloferax volcanii. Here we report a novel small archaeal modifier protein (named SAMP3) with a ß-grasp fold and C-terminal diglycine motif characteristic of ubiquitin that is functional in protein conjugation in Hfx. volcanii. SAMP3 conjugates were dependent on the ubiquitin-activating E1 enzyme homolog of archaea (UbaA) for synthesis and were cleaved by the JAMM/MPN+ domain metalloprotease HvJAMM1. Twenty-three proteins (28 lysine residues) were found to be isopeptide-linked to the C-terminal carboxylate of SAMP3, and 331 proteins were reproducibly found associated with SAMP3 in a UbaA-dependent manner based on tandem mass spectrometry (MS/MS) analysis. The molybdopterin (MPT) synthase large subunit homolog MoaE, found samp3ylated at conserved active site lysine residues in MS/MS analysis, was also shown to be covalently bound to SAMP3 by immunoprecipitation and tandem affinity purifications. HvJAMM1 was demonstrated to catalyze the cleavage of SAMP3 from MoaE, suggesting a mechanism of controlling MPT synthase activity. The levels of samp3ylated proteins and samp3 transcripts were found to be increased by the addition of dimethyl sulfoxide to aerobically growing cells. Thus, we propose a model in which samp3ylation is covalent and reversible and controls the activity of enzymes such as MPT synthase. Sampylation of MPT synthase may govern the levels of molybdenum cofactor available and thus facilitate the scavenging of oxygen prior to the transition to respiration with molybdenum-cofactor-containing terminal reductases that use alternative electron acceptors such as dimethyl sulfoxide. Overall, our study of SAMP3 provides new insight into the diversity of functional ubiquitin-like protein modifiers and the network of ubiquitin-like protein targets in Archaea.

Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii.

BACKGROUND: Thiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms. RESULTS: Based on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant. CONCLUSIONS: Based on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.