Archaea Virtual Special Issue by Wiley-Blackwell

Virtual Special Issue:

Archaea

May 2012

Overview This is a particularly exciting time in Archaeal biology with the development of tools for genetic manipulation and cell biology. Molecular Microbiology continues to publish work that is at the forefront of Archaeal research and ensures a broad audience for the area of Archaeal biology. This virtual special issue showcases articles that have helped guide the field, as well as a selection of empirical papers from recent years that illustrate the quality and depth of the research published in Molecular Microbiology.

Contents Diversity in prokaryotic glycosylation: an archaeal-derived N-linked glycan contains legionaminic acid Lina Kandiba, Olli Aitio, Jari Helin, Ziqiang Guan, Perttu Permi, Dennis H. Bamford, Jerry Eichler and Elina Roine Cdv-based cell division and cell cycle organization in the thaumarchaeon Nitrosopumilus maritimus Erik A. Pelve, Ann-Christin Lindås, Willm Martens-Habbena, José R. de la Torre, David A. Stahl and Rolf Bernander In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon Andrea Manica, Ziga Zebec, Daniela Teichmann and Christa Schleper

Disruption and complementation of the selenocysteine biosynthesis pathway reveals a hierarchy of selenoprotein gene expression in the archaeon Methanococcus maripaludis Tilmann Stock, Mirjam Selzer, Sarah Connery, Deniz Seyhan, Armin Resch and Michael Rother Cover Image – courtesy of Prof. G. Wanner, University of Munich. Cells of Methanocaldococcus villosus adhering to a glass slide. The image is a scanning electron micrograph of superimposed back-scattered and secondary electron signals.

Contents An actin-based cytoskeleton in archaea Thijs J. G. Ettema, Ann-Christin Lindås and Rolf Bernander UV-inducible DNA exchange in hyperthermophilic archaea mediated by type IV pili Małgorzata Ajon, Sabrina Fröls, Marleen van Wolferen, Kilian Stoecker, Daniela Teichmann, Arnold J. M. Driessen, Dennis W. Grogan, Sonja-Verena Albers and Christa Schleper Gene conversion results in the equalization of genome copies in the polyploid haloarchaeon Haloferax volcanii Christian Lange, Karolin Zerulla, Sebastian Breuert and Jörg Soppa Genetic analysis of selenocysteine biosynthesis in the archaeon Methanococcus maripaludis Michael J. Hohn, Sotiria Palioura, Dan Su, Jing Yuan and Dieter Söll Molecular analysis of the crenarchaeal flagellum Kerstin Lassak, Tomasz Neiner, Abhrajyoti Ghosh, Andreas Klingl, Reinhard Wirth and Sonja-Verena Albers

Haloarchaeal myovirus φCh1 harbours a phase variation system for the production of protein variants with distinct cell surface adhesion specificities R. Klein, N. Rössler, M. Iro, H. Scholz and A. Witte Structure–function relationship in an archaebacterial methionine sulphoxide reductase B Michela Carella, Juliane Becher, Oliver Ohlenschläger, Ramadurai Ramachandran, Karl-Heinz Gührs, Gerd Wellenreuther, Wolfram Meyer-Klaucke, Stefan H. Heinemann and Matthias Görlach Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production Thomas J. Santangelo, L'ubomíra Čuboňová and John N. Reeve

Contents Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers Soley Gudbergsdottir, Ling Deng, Zhengjun Chen, Jaide V. K. Jensen, Linda R. Jensen, Qunxin She and Roger A. Garrett In vivo requirement of selenophosphate for selenoprotein synthesis in archaea Tilmann Stock, Mirjam Selzer and Michael Rother

Distinct glycan-charged phosphodolichol carriers are required for the assembly of the pentasaccharide N-linked to the Haloferax volcanii S-layer glycoprotein Ziqiang Guan, Shai Naparstek, Lina Kaminski, Zvia Konrad and Jerry Eichler Regulation of phosphate uptake via Pst transporters in Halobacterium salinarum R1 Katarina Furtw채ngler, Valery Tarasov, Andy Wende, Christoph Schwarz and Dieter Oesterhelt Overlapping repressor binding sites regulate expression of the Methanococcus maripaludis glnK1 operon Thomas J. Lie, Erik L. Hendrickson, Ulf M. Niess, Brian C. Moore, Andrew K. Haydock and John A. Leigh SurR regulates hydrogen production in Pyrococcus furiosus by a sulfur-dependent redox switch Hua Yang, Gina L. Lipscomb, Annette M. Keese, Gerrit J. Schut, Michael Thomm, Michael W. W. Adams, Bi Cheng Wang and Robert A. Scott Methanogenesis by Methanosarcina acetivorans involves two structurally and functionally distinct classes of heterodisulfide reductase Nicole R. Buan and William W. Metcalf Structural insights into the interaction of the crenarchaeal chromatin protein Cren7 with DNA Zhenfeng Zhang, Yong Gong, Li Guo, Tao Jiang and Li Huang

Diversity in prokaryotic glycosylation: an archaeal-derived N-linked glycan contains legionaminic acid

Lina Kandiba, Olli Aitio, Jari Helin, Ziqiang Guan, Perttu Permi, Dennis H. Bamford, Jerry Eichler and Elina Roine Volume 84, Issue 3, May 2012, Pages: 578â&#x20AC;&#x201C;593

Summary VP4, the major structural protein of the haloarchaeal pleomorphic virus, HRPV-1, is glycosylated. To define the glycan structure attached to this protein, oligosaccharides released by Î˛-elimination were analysed by mass spectrometry and nuclear magnetic resonance spectroscopy.

Such analyses showed that the major VP4-derived glycan is a pentasaccharide comprising glucose, glucuronic acid, mannose, sulphated glucuronic acid and a terminal 5-N-formyl-legionaminic acid residue. This is the first observation of legionaminic acid, a sialic acid-like sugar, in an archaeal-derived glycan structure. The importance of this residue for viral infection was demonstrated upon incubation with Nacetylneuraminic acid, a similar monosaccharide. Such treatment reduced progeny virus production by half 4 h post infection. LC-ESI/MS analysis confirmed the presence of pentasaccharide precursors on two different VP4-derived peptides bearing the N-glycosylation signal, NTT. The same sites modified by the native host, Halorubrum sp. strain PV6, were also recognized by the Haloferax volcanii N-glycosylation apparatus, as determined by LC-ESI/MS of heterologously expressed VP4. Here, however, the N-linked pentasaccharide was the same as shown to decorate the S-layer glycoprotein in this species. Hence, N-glycosylation of the haloarchaeal viral protein, VP4, is host-specific. These results thus present additional examples of archaeal N-glycosylation diversity and show the ability of Archaea to modify heterologously expressed proteins.

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Cdv-based cell division and cell cycle organization in the thaumarchaeon Nitrosopumilus maritimus Erik A. Pelve, Ann-Christin Lindås, Willm Martens-Habbena, José R. de la Torre, David A. Stahl and Rolf Bernander Volume 82, Issue 3, November 2011, Pages: 555–566

Summary Cell division is mediated by different mechanisms in different evolutionary lineages. While bacteria and euryarchaea utilize an FtsZ-based mechanism, most crenarchaea divide using the Cdv system, related to the eukaryotic ESCRT-III machinery. Intriguingly, thaumarchaeal genomes encode both FtsZ and Cdv protein homologues, raising the question of their division mode. Here, we provide evidence indicating that Cdv is the primary division system in the thaumarchaeon Nitrosopumilus maritimus. We also show that the cell cycle is differently organized as compared to hyperthermophilic crenarchaea, with a longer pre-replication phase and a shorter post-replication stage. In particular, the time required for chromosome replication is remarkably extensive, 15–18 h, indicating a low replication rate. Further, replication did not continue to termination in a significant fraction of N. maritimus cell populations following substrate depletion. Both the low replication speed and the propensity for replication arrest are likely to represent adaptations to extremely oligotrophic environments. The results demonstrate that thaumarchaea, crenarchaea and euryarchaea display differences not only regarding phylogenetic affiliations and gene content, but also in fundamental cellular and physiological characteristics. The findings also have implications for evolutionary issues concerning the last archaeal common ancestor and the relationship between archaea and eukaryotes.

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In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon Andrea Manica, Ziga Zebec, Daniela Teichmann and Christa Schleper Volume 80, Issue 2, April 2011, Pages: 481–491

Summary Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems are found widespread in bacterial and archaeal genomes and exhibit considerable diversity. However, closer insights into the action of most of the CRISPR modules have remained elusive in particular in Archaea as a result of the lack of suitable in vivo test systems. Here we demonstrate CRISPR/Cas-based immune defence in the hyperthermophilic archaeon Sulfolobus solfataricus. Recombinant variants of the SSV1 virus containing a gene of the conjugative plasmid pNOB8 that represents a target for a corresponding CRISPR spacer in the chromosome were tested in transfection experiments. Almost 100% immunity against the recombinant virus was observed when the chromosomal CRISPR spacer matched perfectly to the protospacer. Different from bacterial systems immunity was still detected, albeit at decreased levels, when mutations distinguished target and spacer. CRISPR/Cas targeting was independent of the transcription of the target gene. Furthermore, a mini-CRISPR locus introduced on the viral DNA with spacers targeting the (non-essential) chromosomal beta-galactosidase gene was unstable in host cells and triggered recombination with the indigenous CRISPR locus. Our experiments demonstrate in vivo activity of CRISPR/Cas in archaea for the first time and suggest that – unlike the recently demonstrated in vitro cleavage of RNA in Pyrococcus– DNA is targeted in this archaeon.

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Summary Proteins containing selenocysteine are found in members of all three domains of life, Bacteria, Eukarya and Archaea. A dedicated tRNA (tRNAsec) serves as a scaffold for selenocysteine synthesis. However, sequence and secondary structures differ in tRNAsec from the different domains.

An Escherichia coli strain lacking the gene for tRNAsec could only be complemented with the homologue from Methanococcus maripaludis when a single base in the anticodon loop was exchanged demonstrating that this base is a crucial determinant for archaeal tRNAsec to function in E. coli. Complementation in trans of M. maripaludis JJ mutants lacking tRNAsec, O-phosphoseryl-tRNAsec kinase or O-phosphoseryl-tRNAsec:selenocysteine synthase with the corresponding genes from M. maripaludis S2 restored the mutant's ability to synthesize selenoproteins. However, only partial restoration of the wild-type selenoproteome was observed as only selenocysteine-containing formate dehydrogenase was synthesized. Quantification of transcripts showed that disrupting the pathway of selenocysteine synthesis leads to downregulation of selenoprotein gene expression, concomitant with upregulation of a seleniumindependent backup system, which is not re-adjusted upon complementation. This transcriptional arrest was independent of selenophosphate but depended on the ‘history’ of the mutants and was inheritable, which suggests that a stable genetic switch may cause the resulting hierarchy of selenoproteins synthesized.

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An actin-based cytoskeleton in archaea Thijs J. G. Ettema, Ann-Christin LindĂĽs and Rolf Bernander Volume 80, Issue 4, May 2011, Pages: 1052â&#x20AC;&#x201C;1061

Summary In eukaryotic and bacterial cells, spatial organization is dependent upon cytoskeletal filaments. Actin is a main eukaryotic cytoskeletal element, involved in key processes such as cell shape determination, mechanical force generation and cytokinesis. We describe an archaeal cytoskeleton which forms helical structures within Pyrobaculum calidifontis cells, as shown by in situ immunostaining. The core components include an archaeal actin homologue, Crenactin, closely related to the eukaryotic counterpart. The crenactin gene belongs to a conserved gene cluster denoted Arcade (actin-related cytoskeleton in Archaea involved in shape determination). The phylogenetic distribution of arcade genes is restricted to the crenarchaeal Thermoproteales lineage, and to Korarchaeota, and correlates with rod-shaped and filamentous cell morphologies. Whereas Arcadin-1, -3 and -4 form helical structures, suggesting cytoskeleton-associated functions, Arcadin-2 was found to be localized between segregated nucleoids in a cell subpopulation, in agreement with possible involvement in cytokinesis. The results support a crenarchaeal origin of the eukaryotic actin cytoskeleton and, as such, have implications for theories concerning the origin of the eukaryotic cell.

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UV-inducible DNA exchange in hyperthermophilic archaea mediated by type IV pili Małgorzata Ajon, Sabrina Fröls, Marleen van Wolferen, Kilian Stoecker, Daniela Teichmann, Arnold J. M. Driessen, Dennis W. Grogan, Sonja-Verena Albers and Christa Schleper Volume 82, Issue 4, November 2011, Pages: 807–817

Summary Archaea, like bacteria and eukaryotes, contain proteins involved in various mechanisms of DNA repair, highlighting the importance of these processes for all forms of life. Species of the order Sulfolobales of hyperthermophilic crenarchaeota are equipped with a strongly UV-inducible type IV pilus system that promotes cellular aggregation. Here we demonstrate by fluorescence in situ hybridization that cellular aggregates are formed based on a species-specific recognition process and that UV-induced cellular aggregation mediates chromosomal marker exchange with high frequency. Recombination rates exceeded those of uninduced cultures by up to three orders of magnitude. Knockout strains of Sulfolobus acidocaldarius incapable of pilus production could not self-aggregate, but were partners in mating experiments with wild-type strains indicating that one cellular partner can mediate the DNA transfer. Since pilus knockout strains showed decreased survival upon UV treatment, we conclude that the UVinducible DNA transfer process and subsequent homologous recombination represents an important mechanism to maintain chromosome integrity in Sulfolobus. It might also contribute substantially to the frequent chromosomal DNA exchange and horizontal gene transfer in these archaea in their natural habitat.

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Gene conversion results in the equalization of genome copies in the polyploid haloarchaeon Haloferax volcanii Christian Lange, Karolin Zerulla, Sebastian Breuert and Jörg Soppa Volume 80, Issue 3, May 2011, Pages: 666–677

Summary Haloferax volcanii is highly polyploid and contains about 20 copies of the major chromosome. A heterozygous strain was constructed that contained two different types of genomes: the leuB locus contained either the wild-type leuB gene or a leuB:trpA gene introduced by gene replacement.

As the trpA locus is devoid of the wild-type trpA gene, growth in the absence of both amino acids is only possible when both types of genomes are simultaneously present, exemplifying gene redundancy and the potential to form heterozygous cells as one possible evolutionary advantage of polyploidy. The heterozygous strain was grown (i) in the presence of tryptophan, selecting for the presence of leuB, (ii) in the presence of leucine selecting for leuB:trpA and (iii) in the absence of selection. Both types of genomes were quantified with real-time PCR. The first condition led to a complete loss of leuB:trpA-containing genomes, while under the second condition leuB-containing genomes were lost. Also in the absence of selection gene conversion led to a fast equalization of genomes and resulted in homozygous leuB-containing cells. Gene conversion leading to genome equalization can explain the escape from ‘Muller's ratchet’ as well as the ease of mutant construction using polyploid haloarchaea.

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Genetic analysis of selenocysteine biosynthesis in the archaeon Methanococcus maripaludis Michael J. Hohn, Sotiria Palioura, Dan Su, Jing Yuan and Dieter SĂśll Volume 81, Issue 1, July 2011, Pages: 249â&#x20AC;&#x201C;258

Summary In Archaea selenocysteine (Sec) is synthesized in three steps. First seryl-tRNA synthetase acylates tRNASec with serine to generate Ser-tRNASec. Then phosphoseryl-tRNASec kinase (PSTK) forms Sep-tRNASec, which is converted to Sec-tRNASec by Sep-tRNA:Sec-tRNA synthase (SepSecS) in the presence of selenophosphate produced by selenophosphate synthetase (SelD). A complete in vivo analysis of the archaeal Sec biosynthesis pathway is still unavailable, and the existence of a redundant pathway or of a rescue mechanism based on the conversion of Sep-tRNASec to Cys-tRNASec during selenium starvation, cannot be excluded. Here we present a mutational analysis of Sec biosynthesis in Methanococcus maripaludis strain Mm900. Sec formation is abolished upon individually deleting the genes encoding SelD, PSTK or SepSecS; the resulting mutant strains could no longer grow on formate while growth with H2 + CO2 remained unaffected. However, deletion of the PSTK and SepSecS genes was not possible unless the selenium-free [NiFe]hydrogenases Frc and Vhc were expressed. This required the prior deletion of either the gene encoding SelD or that of HrsM, a LysR-type regulator suppressing transcription of the frc and vhc operons in the presence of selenium. These results show that M. maripaludis Mm900 is facultatively selenium-dependent with a single pathway of Sec-tRNASec formation.

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Molecular analysis of the crenarchaeal flagellum Kerstin Lassak, Tomasz Neiner, Abhrajyoti Ghosh, Andreas Klingl, Reinhard Wirth and SonjaVerena Albers Volume 83, Issue 1, January 2012, Pages: 110â&#x20AC;&#x201C;124

Summary The ability to move towards favourable conditions provides fundamental advantages to organisms. Interestingly, flagella as motility structures evolved independently in the bacterial and the archaeal kingdom.

Whereas bacterial flagella have been intensively studied, our knowledge regarding the archaeal counterpart is mostly restricted to Euryarchaeota rather than crenarchaeal flagella. We therefore investigated the flagellar assembly system of the crenarchaeal model organism Sulfolobus acidocaldarius in vivo. Promoter studies and qRT-PCR analyses of the flagella gene cluster provided evidence that the expression of the fla genes was induced by tryptone starvation. Moreover, we confirmed presence of a secondary fla promoter within the flaB gene that regulates the transcription of downstream genes flaX-J. Markerless in-frame deletions for all fla genes encoded in the fla gene cluster were constructed. Western blot analysis of all fla deletion strains suggested hierarchical protein interactions during the archaeal flagella assembly. Moreover, functional analysis by thermomicroscopy revealed non-motile cells for each of the mutant strains. Electron micrographs demonstrated that lack of motility coincided with the loss of flagellar assembly. Thus we demonstrated that all seven fla genes are essential for crenarchaeal flagellum assembly and function.

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Summary The φCh1 myovirus, which infects the haloalkaliphilic archaeon Natrialba magadii, contains an invertible region that comprises the convergent open reading frames (ORFs) 34 and 36, which code for the putative tail fibre proteins gp34 and gp36 respectively.

The inversion leads to an exchange of the C-termini of these proteins, thereby creating different types of tail fibres. Gene expression experiments revealed that only ORF34 is transcribed, indicating that φCh1 produces tail fibre proteins exclusively from this particular ORF. Only one of the two types of tail fibres encoded by ORF34 is able to bind to Nab. magadii in vitro. This is reflected by the observation that during the early phases of the infection cycle, the lysogenic strain L11 carries its invertible region exclusively in the orientation that produces that specific type of tail fibre. Obviously, Nab. magadii can only be infected by viruses carrying this particular type of tail fibre. By mutational analysis, the binding domain of gp34 was localized to the C-terminal part of the protein, particularly to a galactose-binding domain. The involvement of galactose residues in cell adhesion was supported by the observation that the addition of α-D-galactose to purified gp34 or whole virions prevented their attachment to Nab. magadii.

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Structure–function relationship in an archaebacterial methionine sulphoxide reductase B Michela Carella, Juliane Becher, Oliver Ohlenschläger, Ramadurai Ramachandran, Karl-Heinz Gührs, Gerd Wellenreuther, Wolfram Meyer-Klaucke, Stefan H. Heinemann and Matthias Görlach Volume 79, Issue 2, January 2011, Pages: 342–358

Summary Oxidation of methionine to methionine sulphoxide (MetSO) may lead to loss of molecular integrity and function. This oxidation can be ‘repaired’ by methionine sulphoxide reductases (MSRs), which reduce MetSO back to methionine.

Two structurally unrelated classes of MSRs, MSRA and MSRB, show stereoselectivity towards the S and the R enantiomer of the sulphoxide respectively. Interestingly, these enzymes were even maintained throughout evolution in anaerobic organisms. Here, the activity and the nuclear magnetic resonance (NMR) structure of MTH711, a zinc containing MSRB from the thermophilic, methanogenic archaebacterium Methanothermobacter thermoautotrophicus, are described. The structure appears more rigid as compared with similar MSRBs from aerobic and mesophilic organisms. No significant structural differences between the oxidized and the reduced MTH711 state can be deduced from our NMR data. A stable sulphenic acid is formed at the catalytic Cys residue upon oxidation of the enzyme with MetSO. The two non-zinc-binding cysteines outside the catalytic centre are not necessary for activity of MTH711 and are not situated close enough to the active-site cysteine to serve in regenerating the active centre via the formation of an intramolecular disulphide bond. These findings imply a reaction cycle that differs from that observed for other MSRBs.

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Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production Thomas J. Santangelo, L'ubomíra Čuboňová and John N. Reeve Volume 81, Issue 4, August 2011, Pages: 897–911

Summary Hydrogen (H2) production by Thermococcus kodakarensis compares very favourably with the levels reported for the most productive algal, fungal and bacterial systems. T. kodakarensis can also consume H2 and is predicted to use several alternative pathways to recycle reduced cofactors, some of which may compete with H2 production for reductant disposal. To explore the reductant flux and possible competition for H2 production in vivo, T. kodakarensis TS517 was mutated to precisely delete each of the alternative pathways of reductant disposal, H2 production and consumption. The results obtained establish that H2 is generated predominantly by the membrane-bound hydrogenase complex (Mbh), confirm the essential role of the SurR (TK1086p) regulator in vivo, delineate the roles of sulfur (S°) regulon proteins and demonstrate that preventing H2 consumption results in a substantial net increase in H2 production. Constitutive expression of TK1086 (surR) from a replicative plasmid restored the ability of T. kodakarensis TS1101 (ΔTK1086) to grow in the absence of S° and stimulated H2 production, revealing a second mechanism to increase H2 production. Transformation of T. kodakarensis TS1101 with plasmids that express SurR variants constructed to direct the constitutive synthesis of the Mbh complex and prevent expression of the S° regulon was only possible in the absence of S° and, under these conditions, the transformants exhibited wild-type growth and H2 production. With S° present, they grew slower but synthesized more H2 per unit biomass than T. kodakarensis TS517.

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Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers Soley Gudbergsdottir, Ling Deng, Zhengjun Chen, Jaide V. K. Jensen, Linda R. Jensen, Qunxin She and Roger A. Garrett Volume 79, Issue 1, January 2011, Pages: 35â&#x20AC;&#x201C;49

Summary The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs.

The genes and protospacers were constructed to carry sequences matching individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers were transcribed. For family I CRISPR loci, the presence of the protospacer CC motif was shown to be important for the occurrence of deletions. The results are consistent with a low level of random dynamic recombination occurring spontaneously, either inter-genomically or intra-genomically, at the repeat regions of Sulfolobus CRISPR loci. Moreover, the relatively high incidence of single-spacer deletions observed for S. islandicus suggests that an additional more directed mechanism operates in this organism.

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In vivo requirement of selenophosphate for selenoprotein synthesis in archaea Tilmann Stock, Mirjam Selzer and Michael Rother Volume 75, Issue 1, January 2010, Pages: 149â&#x20AC;&#x201C;160

Summary Biosynthesis of selenocysteine, the 21st proteinogenic amino acid, occurs bound to a dedicated tRNA in all three domains of life, Bacteria, Eukarya and Archaea, but differences exist between the mechanism employed by bacteria and eukaryotes/archaea.

The role of selenophosphate and the enzyme providing it, selenophosphate synthetase, in archaeal selenoprotein synthesis was addressed by mutational analysis. Surprisingly, MMP0904, encoding a homologue of eukaryal selenophosphate synthetase in Methanococcus maripaludis S2, could not be deleted unless selD, encoding selenophosphate synthetase of Escherichia coli, was present in trans, demonstrating that the factor is essential for the organism. In contrast, the homologous gene of M. maripaludis JJ could be readily deleted, obviating the strain's ability to synthesize selenoproteins. Complementing with selD restored selenoprotein synthesis, demonstrating that the deleted gene encodes selenophosphate synthetase and that selenophosphate is the in vivo selenium donor for selenoprotein synthesis of this organism. We also showed that this enzyme is a selenoprotein itself and that M. maripaludis contains another, HesB-like selenoprotein previously only predicted from genome analyses. The data highlight the use of genetic methods in archaea for a causal analysis of their physiology and, by comparing two closely related strains of the same species, illustrate the evolution of the seleniumutilizing trait.

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Summary In Archaea, dolichol phosphates have been implicated as glycan carriers in the N-glycosylation pathway, much like their eukaryal counterparts. To clarify this relation, highly sensitive liquid chromatography/mass spectrometry was employed to detect and characterize glycan-charged phosphodolichols in the haloarchaeon Haloferax volcanii. It is reported that Hfx. volcanii contains a series of C55 and C60 dolichol phosphates presenting saturated isoprene subunits at the Îą and Ď&#x2030; positions and sequentially modified with the first, second, third and methylated fourth sugar subunits comprising the first four subunits of the pentasaccharide N-linked to the S-layer glycoprotein, a reporter of N-glycosylation. Moreover, when this glycan-charged phosphodolichol pool was examined in cells deleted of agl genes encoding glycosyltransferases participating in N-glycosylation and previously assigned roles in adding pentasaccharide residues one to four, the composition of the lipid-linked glycans was perturbed in the identical manner as was S-layer glycoprotein N-glycosylation in these mutants. In contrast, the fifth sugar of the pentasaccharide, identified as mannose in this study, is added to a distinct dolichol phosphate carrier. This represents the first evidence that in Archaea, as in Eukarya, the oligosaccharides N-linked to glycoproteins are sequentially assembled from glycans originating from distinct phosphodolichol carriers.

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Regulation of phosphate uptake via Pst transporters in Halobacterium salinarum R1 Katarina FurtwĂ¤ngler, Valery Tarasov, Andy Wende, Christoph Schwarz and Dieter Oesterhelt Volume 76, Issue 2, April 2010, Pages: 378â&#x20AC;&#x201C;392

Summary The genome of the archaeon Halobacterium salinarum contains two copies of the pst (phosphate-specific transport) operon, the genes of which are related to well-studied bacterial homologues. Both operons (pst1 and pst2) were shown to be polycistronic and, when under Pi-limited conditions, transcription initiated 1 bp upstream of the translational starts. Under Pi saturation, the pst1 operon utilized an additional transcription start site 59 bp upstream of the first one. The leaderless pst1 transcript was found to be more efficiently translated than the leadered transcript. Promoter strengths differed significantly between the two operons and when Pi levels changed. The basal pst1 promoter activity in Pi-saturated conditions was minimal while the pst2 promoter was active. In contrast, phosphate limitation induced the pst1 operon threefold more than the pst2 operon. We identified basic and phosphate-dependent cis-acting elements in both promoters. Phosphate-uptake assays conducted with several Pst1 and Pst2 mutant strains revealed differences in the substrate affinities between the two transporters and also suggested that the Pi-binding proteins PstS1 and PstS2 can interact with either of the two permease subunits of the transporters. The tactic behaviour of wild type and pst-deletion strains showed that the Pst1 transporter plays an important role for phosphate-directed chemotaxis.

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Overlapping repressor binding sites regulate expression of the Methanococcus maripaludis glnK1 operon Thomas J. Lie, Erik L. Hendrickson, Ulf M. Niess, Brian C. Moore, Andrew K. Haydock and John A. Leigh Volume 75, Issue 3, February 2010, Pages: 755â&#x20AC;&#x201C;762

Summary The euryarchaeal transcriptional repressor NrpR regulates a variety of nitrogen assimilation genes by 2oxoglutarate-reversible binding to conserved palindromic operators. The number and positioning of these operators varies among promoter regions of regulated genes, suggesting NrpR can bind in different patterns. Particularly intriguing is the contrast between the nif and glnK1 promoter regions of Methanococcus maripaludis, where two operators are present but with different configurations. Here we study NrpR binding and regulation at the glnK1 promoter, where the two operator sequences overlap and occur on opposite faces of the double helix. We find that both operators function in binding, with a dimer of NrpR binding simultaneously to each overlapping operator. We show in vivo that the first operator plays a primary role in regulation and the second operator plays an enhancing role. This is the first demonstration of overlapping operators functioning in Archaea.

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SurR regulates hydrogen production in Pyrococcus furiosus by a sulfur-dependent redox switch Hua Yang, Gina L. Lipscomb, Annette M. Keese, Gerrit J. Schut, Michael Thomm, Michael W. W. Adams, Bi Cheng Wang and Robert A. Scott Volume 77, Issue 5, September 2010, Pages: 1111â&#x20AC;&#x201C;1122

Summary We present structural and biochemical evidence for a redox switch in the archaeal transcriptional regulator SurR of Pyrococcus furiosus, a hyperthermophilic anaerobe. P. furiosus produces H2 during fermentation, but undergoes a metabolic shift to produce H2S when elemental sulfur (S0) becomes available. Changes in gene expression occur within minutes of S0 addition, and the majority of these S0-responsive genes are regulatory targets of SurR, a key regulator involved in primary S0 response. SurR was shown in vitro to have dual functionality, activating transcription of some of these genes, notably the hydrogenase operons, and repressing others, including a gene-encoding sulfur reductase. This work demonstrates via biochemical and structural evidence that the activity of SurR is modulated by cysteine residues in a CxxC motif that constitutes a redox switch. Oxidation of the switch with S0 inhibits sequence-specific DNA binding by SurR, leading to deactivation of genes related to H2 production and derepression of genes involved in S0 metabolism.

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Methanogenesis by Methanosarcina acetivorans involves two structurally and functionally distinct classes of heterodisulfide reductase Nicole R. Buan and William W. Metcalf Volume 75, Issue 4, February 2010, Pages: 843â&#x20AC;&#x201C;853

Summary Biochemical studies have revealed two distinct classes of Coenzyme B-Coenzyme M heterodisulfide (CoBS-S-CoM) reductase (Hdr), a key enzyme required for anaerobic respiration in methane-producing archaea.

A cytoplasmic HdrABC enzyme complex is found in most methanogens, whereas a membrane-bound HdrED complex is found exclusively in members of the order Methanosarcinales. Unexpectedly, genomic data indicate that multiple copies of both Hdr classes are found in all sequenced Methanosarcinales genomes. The Methanosarcina acetivorans hdrED1 operon is constitutively expressed and required for viability under all growth conditions examined, consistent with HdrED being the primary Hdr. HdrABC appears to be specifically involved in methylotrophic methanogenesis, based on reduced growth and methanogenesis rates of an hdrA1C1B1 mutant on methylotrophic substrates and downregulation of the genes during growth on acetate. This conclusion is further supported by phylogenetic analysis showing that the presence of hdrA1 in an organism is specifically correlated with the presence of genes for methylotrophic methanogenesis. Examination of mRNA abundance in methanol-grown Î&#x201D;hdrA1C1B1 strains relative to wild-type revealed upregulation of genes required for synthesis of (di)methylsulfide and for transport and biosynthesis of CoB-SH and CoM-SH, suggesting that the mutant has a defect in electron transfer from ferredoxin to CoBS-S-CoM that causes cofactor limitation.

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Structural insights into the interaction of the crenarchaeal chromatin protein Cren7 with DNA Zhenfeng Zhang, Yong Gong, Li Guo, Tao Jiang and Li Huang Volume 76, Issue 3, May 2010, Pages: 749–759

Summary Cren7, a newly found chromatin protein, is highly conserved in the Crenarchaeota. The protein shows higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils in vitro and is associated with genomic DNA in vivo. Here we report the crystal structures of the Cren7 protein from Sulfolobus solfataricus in complex with two DNA sequences. Cren7 binds in the minor groove of DNA and causes a single-step sharp kink in DNA (∼53°) through the intercalation of the hydrophobic side chain of Leu28. Loop β3-β4 of Cren7 undergoes a significant conformational change upon binding of the protein to DNA, suggesting its critical role in the stabilization of the protein–DNA complex. The roles of DNA-contacting amino acid residues in stabilizing the Cren7–DNA interaction were examined by mutational analysis. Structural comparison of Cren7-DNA complexes with Sac7d-DNA complexes reveals significant differences between the two proteins in DNA binding surface, suggesting that Cren7 and Sul7d serve distinct functions in chromosomal organization.

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