Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans. Together, they form an intimate association, the first among Archaea. Membranes are of fundamental importance for the interaction of I. hospitalis and N. equitans, as they harbour the proteins necessary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospitalis cells, and were able to identify 20 proteins in total. Experimental evidence and predictions let us conclude that 11 are soluble cytosolic proteins, eight membrane or membrane-associated proteins, and a single one extracellular. The quantitatively dominating proteins in the cytoplasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells are exposed to at optimal growth conditions. Three abundant membrane protein complexes are found: the major protein of the outer membrane, which might protect the cell against the hostile environment, forms oligomeric complexes with pores of unknown selectivity; two other complexes of the cytoplasmic membrane, the hydrogenase and the ATP synthase, play a key role in energy production and conversion.     

The dominating outer membrane protein of the hyperthermophilic Archaeum Ignicoccus hospitalis: a novel pore-forming complex

The membrane protein Imp1227 (Ignicoccus outer membrane protein; Imp1227) is the main protein constituent of the unique outer sheath of the hyperthermophilic, chemolithoautotrophic Archaeum Ignicoccus hospitalis. This outer sheath is the so far only known example for an asymmetric bilayer among the Archaea and is named 'outer membrane'. With its molecular mass of only 6.23 kDa, Imp1227 is found to be incorporated into the outer membrane in form of large, stable complexes. When separated by SDS-PAGE, they exhibit apparent masses of about 150, 50, 45 and 35 kDa. Dissociation into the monomeric form is achieved by treatment with SDS-containing solutions at temperatures at or above 113 degrees C. Electron micrographs of negatively stained samples confirm that isolated membranes are tightly packed with round complexes, about 7 nm in diameter, with a central, stain-filled 2 nm pore; a local two-dimensional crystalline arrangement in form of small patches can be detected by tomographic reconstruction. The comparison of the nucleotide and amino acid sequence of Imp1227 with public databases showed no reliable similarities with known proteins. Using secondary structure prediction and molecular modelling, an alpha-helical transmembrane domain is proposed; for the oligomer, a ring-shaped nonamer with a central 2 nm pore is a likely arrangement.     

AMP-forming acetyl coenzyme A synthetase in the outermost membrane of the hyperthermophilic crenarchaeon Ignicoccus hospitalis

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic crenarchaeon was found to possess a new CO(2) fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl coenzyme A (acetyl-CoA), which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by two-dimensional native PAGE/SDS-PAGE, followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry and N-terminal sequencing. The ACS of I. hospitalis exhibits a molecular mass of ～690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immunolabeling on cells of I. hospitalis and other described Ignicoccus species revealed that the ACS is localized at the outermost membrane. This perfectly coincides with recent results that the ATP synthase and the H(2):sulfur oxidoreductase complexes are also located in the outermost membrane of I. hospitalis. These results imply that the intermembrane compartment of I. hospitalis is not only the site of ATP synthesis but may also be involved in the primary steps of CO(2) fixation.     

The Iho670 Fibers of Ignicoccus hospitalis: a New Type of Archaeal Cell Surface Appendage

Ignicoccus hospitalis forms many cell surface appendages, the Iho670 fibers (width, 14 nm; length, up to 20 μm), which constitute up to 5% of cellular protein. They are composed mainly of protein Iho670, possessing no homology to archaeal flagellins or fimbrins. Their existence as structures different from archaeal flagella or fimbriae have gone unnoticed up to now because they are very brittle.The existence of surface appendages on archaeal cells has been known for a long time (6; for a recent review, see reference 15); functional studies defined archaeal flagella as motility organelles (e.g., see references 1 and 2) and archaeal fimbriae as adhesins (3, 7, 22). Two other types of very special archaeal cell surface appendages are the hami formed by the SM1 euryarchaeum (13) and the cannulae produced by Pyrodictium occultum (16, 21). In the case of hami, their function is obvious: they are 1- to 3-μm-long filamentous structures with regular spikes, ending in a hook to resemble in ultrastructure a barbed wire, which ends in a grappling hook (13). Thereby, SM1 cells adhere to each other (and structures in their biotope); SM1, indeed can be harvested from polyethylene nets placed into its natural habitat (cold sulfidic springs) (8). The function of the cannulae is still not known: they are extracellular tubes 25 nm wide with an inner diameter of ca. 20 nm and are composed of five related proteins (12). Cannulae enter the periplasmic space but do not reach into the cytoplasm; they have different lengths and connect the highly irregular Pyrodictium cells to form nets easily visible by naked eye. Here, we describe a new type of archaeal cell surface appendage, the fibers which we have detected on cells of the crenarchaeum Ignicoccus hospitalis KIN4/I^T.I. hospitalis originally was described to possess up to nine flagellum-like appendages, anchored at one pole into the cell (17). In repeated experiments designed to examine possible motility of I. hospitalis, however, we never observed such behavior. For those experiments, we used our thermomicroscope, allowing analyses of cells under anaerobic conditions at 90°C (9). To study these cell surface appendages in more detail, cells were grown at 90°C (in medium described in reference 17) either in 120-ml serum bottles filled with 20 ml medium, an 80:20 H₂-CO₂ gas phase, and with shaking at 100 rpm or in a 300-liter fermentor filled with 250 liters as described previously (10). In initial experiments, the cell surface appendages were removed from cells by shearing, differential centrifugation, and a final CsCl gradient centrifugation (similar to the protocol established for the preparation of flagella from Pyrococcus furiosus [14]). Yields, using this method, however, were very low (data not shown). A breakthrough was our finding that the cell surface appendages are very brittle and that the majority of fibers were removed from the cell surface by normal lab handling (compare Fig. 1A and B). Such manipulations include especially sampling of cells using the syringe-needle method (to transfer the strictly anaerobic archaea) and centrifugations to concentrate cells. Therefore, another strategy was used to isolate the cell surface appendages. To aliquots of the supernatant of the harvest (after overnight centrifugation at 16,000 × g) of cells grown to stationary phase in a 300-liter fermentor—containing the majority of fibers, broken off during the harvest centrifugation—NaCl was added to a 5.8% final concentration and polyethylene glycol 6000 (PEG 6000; Fluka, Sigma-Aldrich, Steinheim, Germany) was added to a final concentration of 10.5%. Precipitation was carried out overnight at 6°C, followed by centrifugation (30 min at 10,000 × g). The pelleted material obtained from a 10-liter aliquot of centrifugation supernatant was dissolved in 8 ml of Millipore-purified water (aqua bidest). After addition of 3.6 g of CsCl, the sample was centrifuged for 48 h (SW60 Ti rotor, 250,000 × g, 4°C in Beckman Optima LE-80K centrifuge) and the resulting band (Fig. ​(Fig.1C)1C) was dialyzed extensively against 5 mM MES buffer (1 mM MgSO₄·7H₂O with 1 mM dithiothreitol; pH 6.0). Yields did increase dramatically: the PEG 6000 precipitation method resulted in at least 100-fold more fiber material than the method used initially. (Shearing from 10 liters of cells which had been concentrated by centrifugation resulted in 10 to 50 μg of fiber protein.) In repeated experiments, the total yield of fiber protein, precipitated from 10 liters of centrifugation supernatant by PEG-NaCl addition, varied from 35 to 45 mg. Since the cell yield of 10 liters of culture was 0.75 g, we estimate that the fiber protein constitutes at least 5% of the cellular protein.Open in a separate windowFIG. 1.Ultrastructural and biochemical analyses of Ignicoccus hospitalis fibers. (A) A 5-μl aliquot of a fermentor culture, grown to stationary phase, was applied onto a carbon-coated grid, negatively stained with 2% uranyl acetate, and analyzed by TEM. The sample was collected using a wide-bore pipette. (B) An aliquot of the same culture as that in panel A was analyzed by the TEM procedure as described for panel A. The sample was collected with a syringe and needle. (C) CsCl gradient (volume, 10 ml) of I. hospitalis fibers concentrated by PEG precipitation from 10 liters of supernatant of a 16,000 × g centrifugation (used to harvest cells from the fully grown fermentor culture). (D) TEM analysis of the CsCl gradient-purified fibers, prepared and negatively stained as for panel A. (E to G) SDS-PAGE analyses of CsCl gradient-purified fibers (12.5% gel, silver stained). (E) Broad-range protein marker (NEB). Sizes in kDa are indicated to the left. (F) Heat-denatured fibers (0.5 μg treated for 15 min at 100°C). (G) Partially heat-denatured fibers (5 μg treated for 3 min at 100°C). (H) Amino acid sequence of the fiber protein. The sequence given in boldface and underlined was obtained by N-terminal protein sequencing and used to identify Igni_0670 as the structural gene coding for the I. hospitalis fibers (NCBI reference sequence NC_009776.1). The size bars in panels A, B, and D are 1 μm each.Biochemical and bioinformatic analyses of the fiber protein indicated the following. The material obtained after PEG 6000 precipitation and CsCl gradient centrifugation consisted of mainly one protein with a mass of ca. 33 kDa, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. ​(Fig.1F).1F). Protein samples were resolved by SDS-PAGE with 12.5% polyacrylamide (11); proteins were stained with Coomassie brilliant blue G250 or via silver staining (4). The same preparation, if not completely denatured by heat treatment, resulted in protein bands of ca. 33, 60, and 120 kDa (with weaker bands of ca. 90 and 180 kDa) (Fig. ​(Fig.1G).1G). We take this to indicate that these bands represent fiber protein oligomers. The fiber protein seems not to be glycosylated, as indicated by the lack of a positive reaction (data not shown) with periodic acid-Schiff staining (24), although we note that there is no absolute correlation between the presence or absence of glycosylation with a positive or negative periodic acid-Schiff staining. A lack of glycosylation would differentiate the fiber protein from most other archaeal cell surface appendages described as flagellins, for which such a modification is the rule rather than an exception (23). N-terminal sequencing by Edman degradation (performed by the central protein analytic facility of the Biology Department of the University of Regensburg) determined the N terminus over a length of 23 amino acids (Fig. ​(Fig.1H).1H). Since the genome sequence of I. hospitalis is known (18), identification of the fiber protein was possible: it is encoded by I. hospitalis gene Igni_0670. In order to differentiate fiber proteins potentially occurring in other Ignicoccus species (and to comply with the generally accepted rule to name proteins with a three-letter code), we propose to name the fiber protein of I. hospitalis Iho670. The Iho670 protein is processed since the first 7 amino acids are not found in the mature fiber protein. The two programs Flafind and SOSUI indeed predict such a short signal peptide, while other programs (like signalP) indicate a much longer signal peptide of 38 amino acids: similar difficulties with prediction of signal peptides had been observed earlier for archaeal cell surface proteins, e.g., for the Mth60 fimbrin and the I. hospitalis outer membrane protein Ihomp1 (5, 22; see also reference 3 for a detailed discussion of archaeal signal peptides). A signal peptidase processing the Iho670 protein at the correct site (i.e., after amino acid 7) very recently was identified in I. hospitalis (S.-V. Albers, personal communication). The fiber protein Iho670 shows no homologies to other proteins in its amino acid sequence, especially to the archaeal cell surface appendage proteins identified up to now: archaeal flagellins, the archaeal fimbrin Mth60, the hamus protein, and the three cannula proteins. The Igni_0670 gene might be argued to be part of an operon, because Igni_0668 to Igni_0677 are predicted to be transcribed counterclockwise from the I. hospitalis genome, with a maximum intergenic space of 73 nucleotides (18). Predictions of the proteins encoded by these genes, however, do not favor this possibility, because Igni_0668, Igni_0669, Igni_0670, and Igni_0672 code for hypothetical proteins; Igni_0671 and Igni_0673 code for flavin adenine dinucleotide-dependent pyridine nucleotide disulfide oxidoreductases; Igni_0674 codes for an NiFe hydrogenase maturation protein; Igni_0675 encodes a nonspecific serine/threonine protein kinase; Igni_0676 encodes a protein homologous to eukaryotic initiation factor 1A; and Igni_0677 encodes a 30S ribosomal protein, S6e. Obviously, there is no functional context between the encoded proteins.The results of our ultrastructural analyses of the fibers can be summarized as follows. Transmission electron microscopic (TEM) analyses of the purified Iho670 fibers indicate that they can be up to 20 μm long, with a diameter of 14 nm. TEM analyses of I. hospitalis cells growing on carbon-coated grids (see reference 14 for technical details) confirmed these data. Obviously, these cell surface appendages are very long and brittle; therefore, we were not able to decide how many fibers are synthesized per cell: we estimate this number to be at least 20. For TEM, a drop of cell-suspension was placed on a carbon-coated 200-mesh copper grid (Plano, Wetzlar, Germany). These samples were either unidirectionally shadowed with platinum and carbon at 15° (CFE 50; Cressington Ltd., Watford, United Kingdom) or negatively stained for 1 min with 2% uranyl acetate. All TEM micrographs were recorded using a slow-scan charge-coupled device camera (TEM 1000; TVIPS-Tietz, Gauting, Germany) attached to a CM 12 transmission electron microsope (FEI, Eindhoven, The Netherlands).It turned out that scanning electron microscopic (SEM) analyses of the coccoid cells with emanating fibers is extremely difficult. This is due to the fact that the outer membrane of I. hospitalis is a very delicate structure, being destroyed on nearly every cell during standard fixation and processing steps for TEM and SEM. We have proven that under the same conditions, other archaeal cells and their appendages are well preserved and can be nicely visualized (13, 14, 17, 19, 20). TEM analyses of I. hospitalis cells need special precautions to conserve the labile membranes, like growth in cellulose capillaries and high-pressure freezing and freeze-substitution (19); a simple glutaraldehyde fixation and dehydration at room-temperature will destroy, especially, the outer membrane. For SEM analyses, a protocol preserving the outer membrane is not yet available.     

Filaments from Ignicoccus hospitalis Show Diversity of Packing in Proteins Containing N-Terminal Type IV Pilin Helices

Bacterial motility is driven by the rotation of flagellar filaments that supercoil. The supercoiling involves the switching of coiled-coil protofilaments between two different states. In archaea, the flagellar filaments responsible for motility are formed by proteins with distinct homology in their N-terminal portion to bacterial Type IV pilins. The bacterial pilins have a single N-terminal hydrophobic α-helix, not the coiled coil found in flagellin. We have used electron cryo-microscopy to study the adhesion filaments from the archaeon Ignicoccus hospitalis. While I. hospitalis is non-motile, these filaments make transitions between rigid stretches and curved regions and appear morphologically similar to true archaeal flagellar filaments. A resolution of ~ 7.5 Å allows us to unambiguously build a model for the packing of these N-terminal α-helices, and this packing is different from several bacterial Type IV pili whose structure has been analyzed by electron microscopy and modeling. Our results show that the mechanism responsible for the supercoiling of bacterial flagellar filaments cannot apply to archaeal filaments.     

Properties of the alpha subunit of a Chaperonin from the hyperthermophilic Crenarchaeon Aeropyrum pernix K1

The gene encoding for a putative thermosome from the hyperthermophilic crenarchaeon Aeropyrum pernix K1 (ApcpnA) was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE0907) from A. pernix K1 showed some homology with other group II chaperonins from archaea. The recombinant ApcpnA protein has a molecular mass of 60 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and exhibited ATPase activity with an optimum temperature and pH of 90 degrees C and 5.0, respectively. The ATPase activity was found to be dependent on manganese and potassium ions, but not magnesium ion. The K(m) for ATP at pH 5.0 and 90 degrees C was 10.04 (+/- 1.31) microM, and k(cat) was determined to be 2.21 (+/- 0.11) min(-1) for the ApcpnA monomer. The recombinant ApcpnA prevents thermal aggregation of bovine rhodanese and enhances the thermal stability of alcohol dehydrogenase in vitro, indicating that the protein is suitable as a molecular chaperonin in the high-temperature environment.     

Nanoarchaeum equitans and Ignicoccus hospitalis: new insights into a unique, intimate association of two archaea

Nanoarchaeum equitans and Ignicoccus hospitalis represent a unique, intimate association of two archaea. Both form a stable coculture which is mandatory for N. equitans but not for the host I. hospitalis. Here, we investigated interactions and mutual influence between these microorganisms. Fermentation studies revealed that during exponential growth only about 25% of I. hospitalis cells are occupied by N. equitans cells (one to three cells). The latter strongly proliferate in the stationary phase of I. hospitalis, until 80 to 90% of the I. hospitalis cells carry around 10 N. equitans cells. Furthermore, the expulsion of H2S, the major metabolic end product of I. hospitalis, by strong gas stripping yields huge amounts of free N. equitans cells. N. equitans had no influence on the doubling times, final cell concentrations, and growth temperature, pH, or salt concentration ranges or optima of I. hospitalis. However, isolation studies using optical tweezers revealed that infection with N. equitans inhibited the proliferation of individual I. hospitalis cells. This inhibition might be caused by deprivation of the host of cell components like amino acids, as demonstrated by 13C-labeling studies. The strong dependence of N. equitans on I. hospitalis was affirmed by live-dead staining and electron microscopic analyses, which indicated a tight physiological and structural connection between the two microorganisms. No alternative hosts, including other Ignicoccus species, were accepted by N. equitans. In summary, the data show a highly specialized association of N. equitans and I. hospitalis which so far cannot be assigned to a classical symbiosis, commensalism, or parasitism.     

Insights into the autotrophic CO2 fixation pathway of the archaeon Ignicoccus hospitalis: comprehensive analysis of the central carbon metabolism

Ignicoccus hospitalis is an autotrophic hyperthermophilic archaeon that serves as a host for another parasitic/symbiotic archaeon, Nanoarchaeum equitans. In this study, the biosynthetic pathways of I. hospitalis were investigated by in vitro enzymatic analyses, in vivo (13)C-labeling experiments, and genomic analyses. Our results suggest the operation of a so far unknown pathway of autotrophic CO(2) fixation that starts from acetyl-coenzyme A (CoA). The cyclic regeneration of acetyl-CoA, the primary CO(2) acceptor molecule, has not been clarified yet. In essence, acetyl-CoA is converted into pyruvate via reductive carboxylation by pyruvate-ferredoxin oxidoreductase. Pyruvate-water dikinase converts pyruvate into phosphoenolpyruvate (PEP), which is carboxylated to oxaloacetate by PEP carboxylase. An incomplete citric acid cycle is operating: citrate is synthesized from oxaloacetate and acetyl-CoA by a (re)-specific citrate synthase, whereas a 2-oxoglutarate-oxidizing enzyme is lacking. Further investigations revealed that several special biosynthetic pathways that have recently been described for various archaea are operating. Isoleucine is synthesized via the uncommon citramalate pathway and lysine via the alpha-aminoadipate pathway. Gluconeogenesis is achieved via a reverse Embden-Meyerhof pathway using a novel type of fructose 1,6-bisphosphate aldolase. Pentosephosphates are formed from hexosephosphates via the suggested ribulose-monophosphate pathway, whereby formaldehyde is released from C-1 of hexose. The organism may not contain any sugar-metabolizing pathway. This comprehensive analysis of the central carbon metabolism of I. hospitalis revealed further evidence for the unexpected and unexplored diversity of metabolic pathways within the (hyperthermophilic) archaea.     

Reverse gyrase: an unusual DNA manipulator of hyperthermophilic organisms

Reverse gyrase is the only DNA topoisomerase capable of introducing positive supercoiling into DNA molecules. This unique activity reflects a distinctive arrangement of the protein, which is composed of a topoisomerase IA module fused to a domain containing sequence motives typical of helicases; however, reverse gyrase works neither like a canonical topoisomerase IA nor like a helicase. Extensive genomic analysis has shown that reverse gyrase is present in all organisms living above 70 degrees C and in some of those living at 60- 70 degrees C, but is invariably absent in organisms living at mesophilic temperatures. For its peculiar distribution and biochemical activity, the enzyme has been suggested to play a role in maintenance of genome stability at high temperature. We review here recent phylogenetic, biochemical and structural data on reverse gyrase and discuss the possible role of this enzyme in the biology of hyperthermophilic organisms.     

Molecular biology of extremophiles: recent progress on the hyperthermophilic archaeon Sulfolobus

Extremophiles are microorganisms that flourish in habitats of extreme temperature, pH, salinity, or pressure. All extreme environments are dominated by microorganisms belonging to Archaea, the third domain of life, evolutionary distinct from Bacteria and Eucarya. Over the past few years the biology of extremophilic Archaea has stimulated a lot of interest, aimed at understanding at molecular level the adaptation to their life conditions, as well as their evolutionary relationships to other organisms. Here, we review recent insights in the molecular biology of thermoacidophilic Archaea of the genus Sulfolobus, which has been used as a model system for biochemical, structural, and genetic studies in Archaea and extremophiles in general. With the recent completion of the genome sequence of Sulfolobus solfataricus it is expected that these organisms will contribute new discoveries in the near future. This revised version was published online in August 2006 with corrections to the Cover Date.     

The cell biology of glycosphingolipids

Glycosphingolipids, a family of heterogeneous lipids with biophysical properties conserved from fungi to mammals, are key components of cellular membranes. Because of their tightly packed backbone, they have the ability to associate with other sphingolipids and cholesterol to form microdomains called lipid rafts, with which a variety of proteins associate. These microdomains are thought to originate in the Golgi apparatus, where most sphingolipids are synthesized, and are enriched at the plasma membrane. They are involved in an increasing number of processes, including sorting of proteins by allowing selectivity in intracellular membrane transport. Apart from being involved in recognition and signaling on the cell surface, glycosphingolipids may fulfill unexpected roles on the cytosolic surface of cellular membranes.     

The cell biology of smell

The olfactory system detects and discriminates myriad chemical structures across a wide range of concentrations. To meet this task, the system utilizes a large family of G protein-coupled receptors-the odorant receptors-which are the chemical sensors underlying the perception of smell. Interestingly, the odorant receptors are also involved in a number of developmental decisions, including the regulation of their own expression and the patterning of the olfactory sensory neurons' synaptic connections in the brain. This review will focus on the diverse roles of the odorant receptor in the function and development of the olfactory system.