SummaryArchaea, 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 β-grasp fold and C-terminal diglycine motif similar to ubiquitin, that form protein-conjugates in the archaeon Haloferax volcanii. 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 ε-amino group of lysines from a number of protein targets and Lys58 of SAMP2 itself, revealing poly-SAMP chains. The widespread distribution and diversity of pathways modified by SAMPylation suggest this type of protein-conjugation is central to the archaeal lineage.

Background Haloferax volcanii is an easily culturable moderate halophile that grows on simple defined media, is readily transformable, and has a relatively stable genome. This, in combination with its biochemical and genetic tractability, has made Hfx. volcanii a key model organism, not only for the study of halophilicity, but also for archaeal biology in general.Methodology/Principal FindingsWe report here the sequencing and analysis of the genome of Hfx. volcanii DS2, the type strain of this species. The genome contains a main 2.848 Mb chromosome, three smaller chromosomes pHV1, 3, 4 (85, 438, 636 kb, respectively) and the pHV2 plasmid (6.4 kb).Conclusions/SignificanceThe completed genome sequence, presented here, provides an invaluable tool for further in vivo and in vitro studies of Hfx. volcanii.

Based on our recent work with Haloferax volcanii, ubiquitin-like (Ubl) proteins (SAMP1 and SAMP2) are known to be covalently attached to proteins in archaea. Here, we investigated the enzymes required for the formation of these Ubl-protein conjugates (SAMPylation) and whether this system is linked to sulfur transfer. Markerless in-frame deletions were generated in H. volcanii target genes. The mutants were examined for: (i) the formation of Ubl protein conjugates, (ii) growth under various conditions, including those requiring the synthesis of the sulfur-containing molybdenum cofactor (MoCo), and (iii) the thiolation of tRNA. With this approach we found that UbaA of the E1/MoeB/ThiF superfamily was required for the formation of both SAMP1-and SAMP2-protein conjugates. In addition, UbaA, SAMP1, and MoaE (a homolog of the large subunit of molybdopterin synthase) were essential for MoCo-dependent dimethyl sulfoxide reductase activity, suggesting that these proteins function in MoCo-biosynthesis. UbaA and SAMP2 were also crucial for optimal growth at high temperature and the thiolation of tRNA. Based on these results, we propose a working model for archaea in which the E1-like UbaA can activate multiple Ubl SAMPs for protein conjugation as well as for sulfur transfer. In sulfur transfer, SAMP1 and SAMP2 appear specific for MoCo biosynthesis and the thiolation of tRNA, respectively. Overall, this study provides a fundamental insight into the diverse cellular functions of the Ubl system. posttranslational modification | tRNA modification | proteasomes

Highlights d Discovery of prokaryotic ribosome-associated quality control (RQC) with origin in LUCA d Bacterial RQC is partially redundant with ssrA/tmRNA d Bacterial Rqc2 homolog extends substrates with C-terminal Ala tails that act as degrons d Bacterial RQC protects cells against translation inhibition and environmental stresses

Escherichia coli mutants with defined mutations in specific mod genes that affect molybdate transport were isolated and analyzed for the effects of particular mutations on the regulation of the mod operon as well as the fdhF and hyc operons which code for the components of the formate hydrogenlyase (FHL) complex. ⌽(hyclacZ ؉ ) mod double mutants produced ␤-galactosidase activity only when they were cultured in medium supplemented with molybdate. This requirement was specific for molybdate and was independent of the moa, mob, and moe gene products needed for molybdopterin guanine dinucleotide (MGD) synthesis, as well as Mog protein. The concentration of molybdate required for FHL production by mod mutants was dependent on medium composition. In low-sulfur medium, the amount of molybdate needed by mod mutants for the production of half-maximal FHL activity was increased approximately 20 times by the addition of 40 mM of sulfate. mod mutants growing in low-sulfur medium transported molybdate through the sulfate transport system, as seen by the requirement of the cysA gene product for this transport. In wild-type E. coli, the mod operon is expressed at very low levels, and a mod ؉ merodiploid E. coli carrying a modA-lacZ fusion produced less than 20 units of ␤-galactosidase activity. This level was increased by over 175 times by a mutation in the modA, modB, or modC gene. The addition of molybdate to the growth medium of a mod mutant lowered ⌽(modA-lacZ ؉ ) expression. Repression of the mod operon was sensitive to molybdate but was insensitive to mutations in the MGD synthetic pathway. These physiological and genetic experiments show that molybdate can be transported by one of the following three anion transport systems in E. coli: the native system, the sulfate transport system (cysTWA gene products), and an undefined transporter. Upon entering the cytoplasm, molybdate branches out to mod regulation, fdhF and hyc activation, and metabolic conversion, leading to MGD synthesis and active molybdoenzyme synthesis. Synthesis of active molybdoenzymes byEscherichia coli is dependent on the various gene products required for the transport of molybdate into cells (5,9,12,25,29,39,40,43), metabolic conversion of molybdate to an appropriate form, and incorporation into the pterin component of molybdopterin guanine dinucleotide (MGD) during maturation of the apoprotein to active enzyme (6,8,34,46; see references 13, 38, and 46 for reviews). Several mutant strains of E. coli which are defective in one or more of these steps have been isolated and described. Mutants defective in the first step of this process, molybdate transport, were isolated as chlorate-resistant derivatives (chlD [renamed as mod]) (41) whose phenotype can be suppressed by supplementation of the growth medium with molybdate (5,9,12,13,29,40,43,45). The rates of molybdate transport and internal molybdate concentrations of some of these mutants were determined (5, 12, 40). Recent studies have established that the mod operon contains four genes, of which three r...