Hyperthermophilic Prokaryotes Research Articles

Topological Plasticity of Enzymes Involved in Disulfide Bond Formation Allows Catalysis in Either the Periplasm Or the Cytoplasm

The transmembrane enzymes disulfide bond forming enzyme B (DsbB) and vitamin K epoxide reductase (VKOR) are central to oxidative protein folding in the periplasm of prokaryotes. Catalyzed formation of structural disulfide bonds in proteins also occurs in the cytoplasm of some hyperthermophilic prokaryotes through currently, poorly defined mechanisms. We aimed to determine whether DsbB and VKOR can be inverted in the membrane with retention of activity. By rational design of inversion of membrane topology, we engineered DsbB mutants that catalyze disulfide bond formation in the cytoplasm of Escherichia coli. This represents the first engineered inversion of a transmembrane protein with demonstrated conservation of activity and substrate specificity. This successful designed engineering led us to identify two naturally occurring and oppositely oriented VKOR homologues from the hyperthermophile Aeropyrum pernix that promote oxidative protein folding in the periplasm or cytoplasm, respectively, and hence defines the probable route for disulfide bond formation in the cytoplasm of hyperthermophiles. Our findings demonstrate how knowledge on the determinants of membrane protein topology can be used to de novo engineer a metabolic pathway and to unravel an intriguingly simple evolutionary scenario where a new “adaptive” cellular process is constructed by means of membrane protein topology inversion.

Raman spectra of osmotic solutes of halophiles

Organic osmotic solutes (compatible solutes) are accumulated within the cytoplasm of many microorganisms living in hypersaline environments to provide osmotic balance and to protect the cells against extreme osmotic pressure. Some hyperthermophilic prokaryotes also contain high intracellular concentrations of such compounds. A great diversity of organic osmotic solutes, including small sugars, sugar alcohols, amino acids, and amino acid derivatives, is found in nature. Thanks to the high concentrations in which these compounds are often present, Raman spectroscopy may be a useful technique for rapid analysis of such solute(s) both in individual organisms and in natural microbial communities in high‐salt environments, including the possible use of portable miniaturised Raman spectrometers outdoors. Here, we present a database of Raman spectra of some of the most commonly encountered compatible solutes, as well as some less common ones such as ectoine, hydroxyectoine, glycine betaine, glucosylglycerol, mannosylglycerate (potassium salt), and di‐myo‐inositol phosphate, complementing existing information on the Raman spectra of other such solutes such as glycerol, sucrose, and trehalose. Spectra were collected using excitation at 785 nm, and assignment of the major bands was proposed. The data presented complement our knowledge of the spectroscopic characteristics of biomolecules and enable the rapid assessment of the mode of osmotic adaptation used by halophilic microorganisms in culture; this information may be used to obtain information on the distribution of the different solutes in extreme environments on earth and has the added potential for astrobiological applications to estimate the presence of such solutes in stressed environments elsewhere in the universe. Copyright © 2011 John Wiley & Sons, Ltd.

Structural Analysis of Thermus thermophilus HB27 Mannosyl-3-phosphoglycerate Synthase Provides Evidence for a Second Catalytic Metal Ion and New Insight into the Retaining Mechanism of Glycosyltransferases

Mannosyl-3-phosphoglycerate synthase is a glycosyltransferase involved in the two-step synthetic pathway of mannosylglycerate, a compatible solute that accumulates in response to salt and/or heat stresses in many microorganisms thriving in hot environments. The three-dimensional structure of mannosyl-3-phosphoglycerate synthase from Thermus thermophilus HB27 in its binary complex form, with GDP-alpha-D-mannose and Mg(2+), shows a second metal binding site, about 6 A away from the mannose moiety. Kinetic and mutagenesis studies have shown that this metal site plays a role in catalysis. Additionally, Asp(167) in the DXD motif is found within van der Waals contact distance of the C1' atom in the mannopyranose ring, suggesting its action as a catalytic nucleophile, either in the formation of a glycosyl-enzyme intermediate according to the double-displacement S(N)2 reaction mechanism or in the stabilization of the oxocarbenium ion-like intermediate according to the D(N)*A(Nss) (S(N)i-like) reaction mechanism. We propose that either mechanism may occur in retaining glycosyltransferases with a GT-A fold, and, based on the gathered structural information, we identified an extended structural signature toward a common scaffold between the inverting and retaining glycosyltransferases.

Design of new enzyme stabilizers inspired by glycosides of hyperthermophilic microorganisms

In response to stressful conditions like supra-optimal salinity in the growth medium or temperature, many microorganisms accumulate low-molecular-mass organic compounds known as compatible solutes. In contrast with mesophiles that accumulate neutral or zwitterionic compounds, the solutes of hyperthermophiles are typically negatively charged. (2 R)-2-(α- d-Mannopyranosyl)glycerate (herein abbreviated as mannosylglycerate) is one of the most widespread solutes among thermophilic and hyperthermophilic prokaryotes. In this work, several molecules chemically related to mannosylglycerate were synthesized, namely (2 S)-2-(1- O-α- d-mannopyranosyl)propionate, 2-(1- O-α- d-mannopyranosyl)acetate, (2 R)-2-(1- O-α- d-glucopyranosyl)glycerate and 1- O-(2-glyceryl)-α- d-mannopyranoside. The effectiveness of the newly synthesized compounds for the protection of model enzymes against heat-induced denaturation, aggregation and inactivation was evaluated, using differential scanning calorimetry, light scattering and measurements of residual activity. For comparison, the protection induced by natural compatible solutes, either neutral (e.g., trehalose, glycerol, ectoine) or negatively charged (di- myo-inositol-1,3′-phosphate and diglycerol phosphate), was assessed. Phosphate, sulfate, acetate and KCl were also included in the assays to rank the solutes and new compounds in the Hofmeister series. The data demonstrate the superiority of charged organic solutes as thermo-stabilizers of enzymes and strongly support the view that the extent of protein stabilization rendered by those solutes depends clearly on the specific solute/enzyme examined. The relevance of these findings to our knowledge on the mode of action of charged solutes is discussed.

The Last Universal Common Ancestor: emergence, constitution and genetic legacy of an elusive forerunner

BackgroundSince the reclassification of all life forms in three Domains (Archaea, Bacteria, Eukarya), the identity of their alleged forerunner (Last Universal Common Ancestor or LUCA) has been the subject of extensive controversies: progenote or already complex organism, prokaryote or protoeukaryote, thermophile or mesophile, product of a protracted progression from simple replicators to complex cells or born in the cradle of "catalytically closed" entities? We present a critical survey of the topic and suggest a scenario.ResultsLUCA does not appear to have been a simple, primitive, hyperthermophilic prokaryote but rather a complex community of protoeukaryotes with a RNA genome, adapted to a broad range of moderate temperatures, genetically redundant, morphologically and metabolically diverse. LUCA's genetic redundancy predicts loss of paralogous gene copies in divergent lineages to be a significant source of phylogenetic anomalies, i.e. instances where a protein tree departs from the SSU-rRNA genealogy; consequently, horizontal gene transfer may not have the rampant character assumed by many. Examining membrane lipids suggest LUCA had sn1,2 ester fatty acid lipids from which Archaea emerged from the outset as thermophilic by "thermoreduction," with a new type of membrane, composed of sn2,3 ether isoprenoid lipids; this occurred without major enzymatic reconversion. Bacteria emerged by reductive evolution from LUCA and some lineages further acquired extreme thermophily by convergent evolution. This scenario is compatible with the hypothesis that the RNA to DNA transition resulted from different viral invasions as proposed by Forterre. Beyond the controversy opposing "replication first" to metabolism first", the predictive arguments of theories on "catalytic closure" or "compositional heredity" heavily weigh in favour of LUCA's ancestors having emerged as complex, self-replicating entities from which a genetic code arose under natural selection.ConclusionLife was born complex and the LUCA displayed that heritage. It had the "body "of a mesophilic eukaryote well before maturing by endosymbiosis into an organism adapted to an atmosphere rich in oxygen. Abundant indications suggest reductive evolution of this complex and heterogeneous entity towards the "prokaryotic" Domains Archaea and Bacteria. The word "prokaryote" should be abandoned because epistemologically unsound.ReviewersThis article was reviewed by Anthony Poole, Patrick Forterre, and Nicolas Galtier.

Microbial CO2 fixation and sulfur cycling associated with low‐temperature emissions at the Lilliput hydrothermal field, southern Mid‐Atlantic Ridge (9°S)

Lilliput was discovered in 2005 as the southernmost known hydrothermal field along the Mid-Atlantic Ridge. It is exceptional in that it lacks high-temperature venting probably because of a thickened crust. The absence of thermophilic and hyperthermophilic prokaryotes in emissions supports the argument against the presence of a hot subsurface at Lilliput, as is typically suggested for diffuse emissions from areas of high-temperature venting. The high phylogenetic diversity and novelty of bacteria observed could be because of the low-temperature influence, the distinct location of the hydrothermal field or the Bathymodiolus assemblages covering the sites of discharge. The low-temperature fluids at the Lilliput are characterized by lowered pH and slightly elevated hydrogen (16 nM) and methane ( approximately 2.6 microM) contents compared with ambient seawater. No typical hydrogen and methane oxidizing prokaryotes were detected. The higher diversity of reverse tricarboxylic acid genes and the form II RubisCO genes of the Calvin Benson-Bassham (CBB) cycle compared with the form I RubisCO genes of the CBB cycle suggests that the chemoautotrophic community is better adapted to low oxygen concentrations. Thiomicrospira spp. and Epsilonproteobacteria dominated the autotrophic community. Sulfide is the most abundant inorganic energy source (0.5 mM). Diverse bacteria were associated with sulfur cycling, including Gamma-, Delta- and Epsilonproteobacteria, with the latter being the most abundant bacteria according to fluorescence in situ hybridization. With members of various Candidate Divisions constituting for 25% of clone library sequences we suggest that their role in vent ecosystems might be more important than previously assumed and propose potential mechanisms they might be involved in at the Lilliput hydrothermal field.

Studies of Thermophilic Microorganisms at the Institute of Microbiology, Russian Academy of Sciences

The thermophily studies at the Institute of Microbiology (INMI), Russian Academy of Sciences, were started more than six decades back, at the end of the 1930s and in the early 1940s. The first experiments to study the metabolic diversity of thermophiles and their physiology, biochemistry, and enzymology were conducted at the institute by A.A. Imshenetskii and his coworkers L.I. Solntseva, S.Z. Brotskaya, and L.A. Kuzyurina. The obtained experimental evidence relating to cellulolytic, amylolytic, and proteolytic thermophiles and their enzymes was generalized in Imshenetskii’s monograph Microbiological Processes at High Temperatures [1]. This book also compared thermophilic and mesophilic processes and discussed the origin of thermophiles. In 1950, the contemporary view of thermophily was presented in E.N. Mishustin’s monograph Thermophilic Organisms in Nature and Practice [2], which dealt not only with general and specific properties of thermophilic organisms but also considered their habitats and possible applications. Unlike metabolic diversity, the phylogeny of thermophiles did not figure prominently in any of these monographs, and the experiments and conclusions regarding adaptation of mesophilic species to high temperatures should be regarded as a tribute to the ideology prevailing at the time. Dealing with thermophiles in natural environments, the book focused on soil and anthropogenic biotopes (compost, silage, and selfheating substrates) and on the gastrointestinal tract of higher organisms. The pioneering paper on the microbiology of volcanic habitats was published by S.I. Kuznetsov and was based on his studies of Kamchatka hot springs [3]. The conclusions of this paper outlined all major lines of investigations of thermophilic prokaryotes in their natural habitats that a few decades later were being conducted at the Institute of Microbiology. In the work of Kuznetsov, hyperthermophilic prokaryotes growing at temperatures over 90°e were found and thermophilic microorganisms were shown to be functional in the reductive and oxidative parts of the sulfur cycle and to be involved in mineral formation processes in hot springs.

A gene from the mesophilic bacterium Dehalococcoides ethenogenes encodes a novel mannosylglycerate synthase.

Mannosylglycerate (MG) is a common compatible solute found in thermophilic and hyperthermophilic prokaryotes. In this study we characterized a mesophilic and bifunctional mannosylglycerate synthase (MGSD) encoded in the genome of the bacterium Dehalococcoides ethenogenes. mgsD encodes two domains with extensive homology to mannosyl-3-phosphoglycerate synthase (MPGS, EC 2.4.1.217) and to mannosyl-3-phosphoglycerate phosphatase (MPGP, EC 3.1.3.70), which catalyze the consecutive synthesis and dephosphorylation of mannosyl-3-phosphoglycerate to yield MG in Pyrococcus horikoshii, Thermus thermophilus, and Rhodothermus marinus. The bifunctional MGSD was overproduced in Escherichia coli, and we confirmed the combined MPGS and MPGP activities of the recombinant enzyme. The optimum activity of the enzyme was at 50 degrees C. To examine the properties of each catalytic domain of MGSD, we expressed them separately in E. coli. The monofunctional MPGS was unstable, while the MPGP was stable and was characterized. Dehalococcoides ethenogenes cannot be grown sufficiently to identify intracellular compatible solutes, and E. coli harboring MGSD did not accumulate MG. However, Saccharomyces cerevisiae expressing mgsD accumulated MG, confirming that this gene product can synthesize this compatible solute and arguing for a role in osmotic adjustment in the natural host. We did not detect MGSD activity in cell extracts of S. cerevisiae. Here we describe the first gene and enzyme for the synthesis of MG from a mesophilic microorganism and discuss the possible evolution of this bifunctional MGSD by lateral gene transfer from thermophilic and hyperthermophilic organisms.

The Domain Organization and Properties of Individual Domains of DNA Topoisomerase V, a Type 1B Topoisomerase with DNA Repair Activities

Topoisomerase V (Topo V) is a type IB (eukaryotic-like) DNA topoisomerase. It was discovered in the hyperthermophilic prokaryote Methanopyrus kandleri and is the only topoisomerase with associated apurinic/apyrimidinic (AP) site-processing activities. The structure of Topo V in the free and DNA-bound states was probed by limited proteolysis at 37 degrees C and 80 degrees C. The Topo V protein is comprised of (i) a 44-kDa NH(2)-terminal core subdomain, which contains the active site tyrosine residue for topoisomerase activity, (ii) an immediately adjacent 16-kDa subdomain that contains degenerate helix-hairpin-helix (HhH) motifs, (iii) a protease-sensitive 18-kDa HhH "hinge" region, and (iv) a 34-kDa COOH-terminal HhH domain. Three truncated Topo V polypeptides comprising the NH(2)-terminal 44-kDa and 16-kDa domains (Topo61), the 44-, 16-, and 18-kDa domains (Topo78), and the COOH-terminal 34-kDa domain (Topo34) were cloned, purified, and characterized. Both Topo61 and Topo78 are active topoisomerases, but in contrast to Topo V these enzymes are inhibited by high salt concentrations. Topo34 has strong DNA-binding ability but shows no topoisomerase activity. Finally, we demonstrate that Topo78 and Topo34 possess AP lyase activities that are important in base excision DNA repair. Thus, Topo V has at least two active sites capable of processing AP DNA. The significance of multiple HhH motifs for the Topo V processivity is discussed.

Towards the ecology of hyperthermophiles: biotopes, new isolation strategies and novel metabolic properties.

Ecological studies have shown that water-containing terrestrial, subterranean and submarine high-temperature environments harbor a great diversity of hyperthermophilic prokaryotes, growing fastest at temperatures of 80 degrees C or above. The investigations included cultivation, isolation and detailed analysis of these hyperthermophiles as well as in situ 16S rRNA gene sequence analysis and in situ hybridization studies. For a safe and fast isolation of novel hyperthermophiles from mixed cultures, a new, plating-independent isolation technique was developed, based on the use of a laser microscope ('optical tweezers'). This method, combined with 16S rRNA gene sequence analysis and whole-cell hybridization using fluorescently labelled oligonucleotide probes, even allows the recovery of pure cultures of phylogenetically predicted organisms harboring novel 16S rRNA gene sequences. In their natural habitats, hyperthermophiles form complex food webs, consisting of primary producers and consumers of organic material. Their metabolic potential includes various types of aerobic and anaerobic respiration and different modes of fermentation. In hydrothermal and geothermal environments, hyperthermophiles have important ecological functions in biogeochemical processes. Members of the Sulfolobales are able to mobilize heavy metals from sulfidic ores like pyrite or chalcopyrite. Biomineralization processes of hyperthermophiles include the formation of magnetite from iron or the precipitation of arsenate as realgar, a reaction performed by a novel hyperthermophile that was isolated from Pisciarelli Solfatara, Naples, Italy.

Ecologic Genomics of DNA: Upstream Bending in Prokaryotic Promoters

After our analysis of the distribution of predicted intrinsic curvature along all available complete prokaryotic genomes, the genomes were divided into two groups. Curvature distribution in all prokaryotes of the first group indicated a substantial fraction of promoters characterized by intrinsic DNA curvature located within or upstream of the promoter region. We did not find this peculiar DNA curvature distribution in prokaryotes in the second group. Remarkably, all bacteria of the first group were mesophilic, whereas many prokaryotes of the second group were hyperthermophilic. We hypothesize that DNA curvature plays a biologic role in gene regulation in mesophilic as opposed to hyperthermophilic prokaryotes, i.e., DNA curvature presumably has a functional adaptive significance determined by temperature selection.

Reverse Gyrase from Methanopyrus kandleri: RECONSTITUTION OF AN ACTIVE EXTREMOZYME FROM ITS TWO RECOMBINANT SUBUNITS

Reverse gyrases are ATP-dependent type I 5'-topoisomerases that positively supercoil DNA. Reverse gyrase from Methanopyrus kandleri is unique as the first heterodimeric type I 5'-topoisomerase described, consisting of a 138-kDa subunit involved in the hydrolysis of ATP (RgyB) and a 43-kDa subunit that forms the covalent complex with DNA during the topoisomerase reaction (RgyA). Here we report the reconstitution of active reverse gyrase from the two recombinant proteins overexpressed in Escherichia coli. Both proteins have been purified by column chromatography to >90% homogeneity. RgyB has a DNA-dependent ATPase activity at high temperature (80 degrees C) and is independent of the presence of RgyA. RgyA alone has no detectable activity. The addition of RgyA to RgyB reconstitutes positive supercoiling activity, but the RgyB and RgyA subunits form a stable heterodimer only after being heated together. This is the first case in which it has been possible to reconstitute an active heterodimeric enzyme of a hyperthermophilic prokaryote from recombinant proteins.

Purification and characterization of DNA topoisomerase V. An enzyme from the hyperthermophilic prokaryote Methanopyrus kandleri that resembles eukaryotic topoisomerase I.

DNA topoisomerase V is a novel prokaryotic enzyme related to eukaryotic topoisomerase I. The enzyme is a type I DNA topoisomerase and is recognized by polyclonal antibody against human topoisomerase I. We describe its purification from the hyperthermophilic methanogen Methanopyrus kandleri. The enzyme has high activity in crude extracts and is present in at least 1,500 copies/cell. Topoisomerase V migrates as a 110-kDa polypeptide in SDS-polyacrylamide gel electrophoresis and as a 142-kDa globular protein in gel filtration. It is active up to at least 100 degrees C on both positively and negatively supercoiled DNA and is not inhibited by single-stranded DNA. The enzyme works from 1 to 650 mM NaCl and up to 3.1 M potassium glutamate. It acts processively at low ionic strength and distributively at high NaCl or KCl concentration. Magnesium is not required and does not stimulate the enzymatic activity. Under DNA denaturing conditions, topoisomerase V catalyzes an unlinking reaction which results in substantial reduction in the linking number of closed circular DNA. The driving force for this process is DNA melting. Camptothecin is not nearly as good an inhibitor for topoisomerase V as it is for eukaryotic topoisomerase I. The unique occurrence of two major type I topoisomerases (reverse gyrase and topoisomerase V) in M. kandleri may shed new light on the evolution of this family of enzymes and supports the concept of a distant but significant relationship between some hyperthermophilic organisms and eukaryotes.

DNA topoisomerase V is a relative of eukaryotic topoisomerase I from a hyperthermophilic prokaryote.

The DNA topoisomerases are ubiquitous enzymes that fulfil vital roles in the replication, transcription and recombination of DNA by carrying out DNA-strand passage reactions. Here we characterize a prokaryotic counterpart to the eukaryotic topoisomerase I in the hyperthermophilic methanogen Methanopyrus kandleri. The new enzyme, called topoisomerase V, has the following properties in common with eukaryotic topoisomerase I, which distinguish it from all other known prokaryotic topoisomerases: (1) its activity is Mg(2+)-independent; (2) it relaxes both negatively and positively supercoiled DNA; (3) it makes a covalent complex with the 3' end of the broken DNA strand; and (4) it is recognized by antibody raised against human topoisomerase I. Eukaryotic-like enzymes have been discovered in some hyperthermophilic prokaryotes, namely the eocytes and the extremely thermophilic archaebacteria, and hyperthermophilic homologues of eukaryotic DNA polymerase-alpha, transcription factor IIB and DNA ligase have all been reported. Thus our findings support the idea that some essential parts of the eukaryotic transcription-translation and replication machineries were in place before the emergence of eukaryotes, and that the closest living relatives of eukaryotes may be hyperthermophiles.

The nature of the last universal ancestor and the root of the tree of life, still open questions

The nature of the last universal ancestor to all extent cellular organisms and the rooting of the universal tree of life are fundamental questions which can now be adressed by molecular evolutionists. Several scenarios have been proposed during the last years, based on the phylogenies of ribosomal RNA and of duplicated proteins, which suggest that the last universal ancestor was either an RNA progenote or an hyperthermophilic prokaryote. We discuss these hypotheses in the light of new data on the evolution of DNA metabolizing enzymes and of contradictions between different protein phylogenies. We conclude that the last universal ancestor was a member of the DNA world already containing several DNA polymerases and DNA topoisomerases. Furthermore, we criticize current data which suggest that the rooting of the universal tree of life is located in the eubacterial branch and we conclude that both rooting the universal tree and the nature of the last universal ancestor are still open questions.