Agl24 is an ancient archaeal homolog of the eukaryotic N-glycan chitobiose synthesis enzymes.

Protein N-glycosylation is a post-translational modification found in organisms of all domains of life. The crenarchaeal N-glycosylation begins with the synthesis of a lipid-linked chitobiose core structure, identical to that in Eukaryotes, although the enzyme catalyzing this reaction remains unknown. Here, we report the identification of a thermostable archaeal ß-1,4-N-acetylglucosaminyltransferase, named archaeal glycosylation enzyme 24 (Agl24), responsible for the synthesis of the N-glycan chitobiose core. Biochemical characterization confirmed its function as an inverting ß-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase. Substitution of a conserved histidine residue, found also in the eukaryotic and bacterial homologs, demonstrated its functional importance for Agl24. Furthermore, bioinformatics and structural modeling revealed similarities of Agl24 to the eukaryotic Alg14/13 and a distant relation to the bacterial MurG, which are catalyzing the same or a similar reaction, respectively. Phylogenetic analysis of Alg14/13 homologs indicates that they are ancient in Eukaryotes, either as a lateral transfer or inherited through eukaryogenesis.

The missing enzymatic link in syntrophic methane formation from fatty acids.

The microbial production of methane from organic matter is an essential process in the global carbon cycle and an important source of renewable energy. It involves the syntrophic interaction between methanogenic archaea and bacteria that convert primary fermentation products such as fatty acids to the methanogenic substrates acetate, H2, CO2, or formate. While the concept of syntrophic methane formation was developed half a century ago, the highly endergonic reduction of CO2 to methane by electrons derived from ß-oxidation of saturated fatty acids has remained hypothetical. Here, we studied a previously noncharacterized membrane-bound oxidoreductase (EMO) from Syntrophus aciditrophicus containing two heme b cofactors and 8-methylmenaquinone as key redox components of the redox loop-driven reduction of CO2 by acyl-coenzyme A (CoA). Using solubilized EMO and proteoliposomes, we reconstituted the entire electron transfer chain from acyl-CoA to CO2 and identified the transfer from a high- to a low-potential heme b with perfectly adjusted midpoint potentials as key steps in syntrophic fatty acid oxidation. The results close our gap of knowledge in the conversion of biomass into methane and identify EMOs as key players of ß-oxidation in (methyl)menaquinone-containing organisms.

Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme.

Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.

Expanding the archaellum regulatory network - the eukaryotic protein kinases ArnC and ArnD influence motility of Sulfolobus acidocaldarius.

Expression of the archaellum, the archaeal-type IV pilus-like rotating motility structure is upregulated under nutrient limitation. This is controlled by a network of regulators, called the archaellum regulatory network (arn). Several of the components of this network in Sulfolobus acidocaldarius can be phosphorylated, and the deletion of the phosphatase PP2A results in strongly increased motility during starvation, indicating a role for phosphorylation in the regulation of motility. Analysis of the motility of different protein kinase deletion strains revealed that deletion of saci_0965, saci_1181, and saci_1193 resulted in reduced motility, whereas the deletion of saci_1694 resulted in hypermotility. Here ArnC (Saci_1193) and ArnD (Saci_1694) are characterized. Purified ArnC and ArnD phosphorylate serine and threonine residues in the C-terminus of the repressor ArnB. arnC is upregulated in starvation medium, whereas arnD is constitutively expressed. However, while differences in the expression and levels of flaB were observed in the ΔarnD strain during growth under rich conditions, under nutrient limiting conditions the ΔarnC and ΔarnD strains showed no large differences in the expression levels of the archaellum or of the studied regulators. This suggests that next to the regulation via the archaellum regulatory network additional regulatory mechanisms of expression and/or activity of the archaellum exist.

Archaeal TFEα/ß is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39.

Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and ß subunits. Here we have identified and characterised the function of the TFIIEß homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39. Both archaeal TFEß and hRPC39 harbour a cubane 4Fe-4S cluster, which is crucial for heterodimerization of TFEα/ß and its engagement with the RNAP clamp. TFEα/ß stabilises the preinitiation complex, enhances DNA melting, and stimulates abortive and productive transcription. These activities are strictly dependent on the ß subunit and the promoter sequence. Our results suggest that archaeal TFEα/ß is likely to represent the evolutionary ancestor of TFIIE-like factors in extant eukaryotes.

The sub-cellular localization of Sulfolobus DNA replication.

Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.

Chromosome segregation in Archaea mediated by a hybrid DNA partition machine.

Eukarya and, more recently, some bacteria have been shown to rely on a cytoskeleton-based apparatus to drive chromosome segregation. In contrast, the factors and mechanisms underpinning this fundamental process are underexplored in archaea, the third domain of life. Here we establish that the archaeon Sulfolobus solfataricus harbors a hybrid segrosome consisting of two interacting proteins, SegA and SegB, that play a key role in genome segregation in this organism. SegA is an ortholog of bacterial, Walker-type ParA proteins, whereas SegB is an archaea-specific factor lacking sequence identity to either eukaryotic or bacterial proteins, but sharing homology with a cluster of uncharacterized factors conserved in both crenarchaea and euryarchaea, the two major archaeal sub-phyla. We show that SegA is an ATPase that polymerizes in vitro and that SegB is a site-specific DNA-binding protein contacting palindromic sequences located upstream of the segAB cassette. SegB interacts with SegA in the presence of nucleotides and dramatically affects its polymerization dynamics. Our data demonstrate that SegB strongly stimulates SegA polymerization, possibly by promoting SegA nucleation and accelerating polymer growth. Increased expression levels of segAB resulted in severe growth and chromosome segregation defects, including formation of anucleate cells, compact nucleoids confined to one half of the cell compartment and fragmented nucleoids. The overall picture emerging from our findings indicates that the SegAB complex fulfills a crucial function in chromosome segregation and is the prototype of a DNA partition machine widespread across archaea.

The archaeal exosome localizes to the membrane.

We studied the cellular localization of the archaeal exosome, an RNA-processing protein complex containing orthologs of the eukaryotic proteins Rrp41, Rrp42, Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG. Fractionation of cell-free extracts of Sulfolobus solfataricus in sucrose density gradients revealed that DnaG and the active-site comprising subunit Rrp41 are enriched together with surface layer proteins in a yellow colored ring, implicating that the exosome is membrane-bound. In accordance with this assumption, DnaG and Rrp41 were detected at the periphery of the cell by immunofluorescence microscopy. Our finding suggests that RNA processing in Archaea is spatially organized.

"Hot standards" for the thermoacidophilic archaeon Sulfolobus solfataricus.

Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology ("SulfoSYS")-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the "-omics" approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics.

The Sulfolobus solfataricus AAA protein Sso0909, a homologue of the eukaryotic ESCRT Vps4 ATPase.

Sso0909 is a protein of the thermo-acidophilic crenarchaeon Sulfolobus solfataricus, annotated as a p60 katanin-like ATPase. We present here results supporting the hypothesis that Sso0909 is an orthologue of the eukaryotic ESCRT (endosomal sorting complex required for transport) ATPase Vps4 (vacular protein sorting 4). The spectrum of Sso0909 homologues is limited to several orders of Crenarchaea and to three euryarchaeal Thermoplasmata species, where they were presumably acquired by lateral gene transfer. Almost invariably, Sso0909 homologues occur in the genomic vicinity of homologues of eukaryotic ESCRT-III components, which are the targets of disassembly by Vps4, as well as with a creanarchaeal-specific coiled-coil protein. S. solfataricus sso0909 is constitutively expressed under normal growth conditions and appears to be essential, as judged by the failure to obtain stable deletion mutants. We expressed Sso0909 in Escherichia coli and S. solfataricus, but have not obtained preparations with ATPase activity so far.

Identification of diverse archaeal proteins with class III signal peptides cleaved by distinct archaeal prepilin peptidases.

Most secreted archaeal proteins are targeted to the membrane via a tripartite signal composed of a charged N terminus and a hydrophobic domain, followed by a signal peptidase-processing site. Signal peptides of archaeal flagellins, similar to class III signal peptides of bacterial type IV pilins, are distinct in that their processing sites precede the hydrophobic domain, which is crucial for assembly of these extracytoplasmic structures. To identify the complement of archaeal proteins with class III signal sequences, a PERL program (FlaFind) was written. A diverse set of proteins was identified, and many of these FlaFind positives were encoded by genes that were cotranscribed with homologs of pilus assembly genes. Moreover, structural conservation of primary sequences between many FlaFind positives and subunits of bacterial pilus-like structures, which have been shown to be critical for pilin assembly, have been observed. A subset of pilin-like FlaFind positives contained a conserved domain of unknown function (DUF361) within the signal peptide. Many of the genes encoding these proteins were in operons that contained a gene encoding a novel euryarchaeal prepilin-peptidase, EppA, homolog. Heterologous analysis revealed that Methanococcus maripaludis DUF361-containing proteins were specifically processed by the EppA homolog of this archaeon. Conversely, M. maripaludis preflagellins were cleaved only by the archaeal preflagellin peptidase FlaK. Together, the results reveal a diverse set of archaeal proteins with class III signal peptides that might be subunits of as-yet-undescribed cell surface structures, such as archaeal pili.

Functional and structural characterization of the minimal Sec translocase of the hyperthermophile Thermotoga maritima.

The genome of the hyperthermophilic bacterium Thermotoga maritima contains the genes that encode core subunits of the protein translocase, a complex consisting of the molecular motor SecA and the protein conducting pore SecYE. In addition, we identified an erroneous sequence in the genome encoding for a putative secG gene. The genes of the T. maritima translocase subunits were overexpressed in Escherichia coli and purified to homogeneity. T. maritima SecA showed a basal thermostable ATPase activity that was stimulated up to 4-fold by phospholipids with an optimum at 74 degrees C. Membrane vesicles and proteoliposomes containing SecYE or SecYEG supported 2- to 4-fold stimulation of the precursor dependent SecA ATPase activity. Imaging of small two-dimensional crystals of the SecYE complex using electron microscopy showed square-shaped particles with a side-length of about 6 nm. These results demonstrate that in T. maritima a highly thermostable translocase complex is operational.

Crystal structures of the ATPase subunit of the glucose ABC transporter from Sulfolobus solfataricus: nucleotide-free and nucleotide-bound conformations.

The ABC-ATPase GlcV energizes a binding protein-dependent ABC transporter that mediates glucose uptake in Sulfolobus solfataricus. Here, we report high-resolution crystal structures of GlcV in different states along its catalytic cycle: distinct monomeric nucleotide-free states and monomeric complexes with ADP-Mg(2+) as a product-bound state, and with AMPPNP-Mg(2+) as an ATP-like bound state. The structure of GlcV consists of a typical ABC-ATPase domain, comprising two subdomains, connected by a linker region to a C-terminal domain of unknown function. Comparisons of the nucleotide-free and nucleotide-bound structures of GlcV reveal re-orientations of the ABCalpha subdomain and the C-terminal domain relative to the ABCalpha/beta subdomain, and switch-like rearrangements in the P-loop and Q-loop regions. Additionally, large conformational differences are observed between the GlcV structures and those of other ABC-ATPases, further emphasizing the inherent flexibility of these proteins. Notably, a comparison of the monomeric AMPPNP-Mg(2+)-bound GlcV structure with that of the dimeric ATP-Na(+)-bound LolD-E171Q mutant reveals a +/-20 degrees rigid body re-orientation of the ABCalpha subdomain relative to the ABCalpha/beta subdomain, accompanied by a local conformational difference in the Q-loop. We propose that these differences represent conformational changes that may have a role in the mechanism of energy-transduction and/or allosteric control of the ABC-ATPase activity in bacterial importers.

Purification, crystallization and preliminary X-ray diffraction analysis of an archaeal ABC-ATPase.

In the archaeon Sulfolobus solfataricus glucose uptake is mediated by an ABC transport system. The ABC-ATPase of this transporter (GlcV) has been overproduced in Escherichia coli and purified. Crystals of GlcV suitable for data collection were obtained in the absence of nucleotide by microseeding combined with vapour diffusion from a mixture of PEG polymers and NaCl. Appearing under identical conditions, two crystal forms have been characterized by X-ray diffraction. Both forms diffract to high resolution using synchrotron radiation and both belong to space group P2(1)2(1)2(1). The related crystal forms A (unit-cell parameters a = 47.0, b = 48.2, c = 182.1 A) and B (a = 47.0, b = 146.6, c = 178.5 A) feature one and three GlcV molecules in the asymmetric unit, respectively, with a solvent content of about 50%. Crystals have also been obtained in the presence of sodium iodide. From single-wavelength anomalous diffraction data extending to 2.1 A resolution, an iodide substructure could be resolved.