Remarkable morphological diversity of viruses and virus-like particles in hot terrestrial environments.

Electron microscopic studies of the viruses in two hot springs (85 degrees C, pH 1.5-2.0, and 75-93 degrees C, pH 6.5) in Yellowstone National Park revealed particles with twelve different morphotypes. This diversity encompassed known viruses of hyperthermophilic archaea, filamentous Lipothrixviridae, rod-shaped Rudiviridae, and spindle-shaped Fuselloviridae, and novel morphotypes previously not observed in nature. Two virus types resembled head-and-tail bacteriophages from the families Siphoviridae and Podoviridae, and constituted the first observation of these viruses in a hydrothermal environment. Viral hosts in the acidic spring were members of the hyperthermophilic archaeal genus Acidianus.

Cytochromes c555 from the hyperthermophilic bacterium Aquifex aeolicus. 2. Heterologous production of soluble cytochrome c555s and investigation of the role of methionine residues.

The cycB2 gene encoding the soluble cytochrome c555s from Aquifex aeolicus, an hyperthermophilic organism, has been cloned and expressed using Escherichia coli as the host organism. The cytochrome was successfully produced in the periplasm of an E. coli strain coexpressing the ccmABCDEFGH genes involved in the cytochrome c maturation process. Comparison of native and recombinant cytochrome c555s shows that both proteins are indistinguishable in terms of spectroscopic and physicochemical properties. Since two different methionine residues are present in the sequence stretch usually providing the sixth ligand to the heme iron, site-directed mutagenesis has been performed in order to identify the methionine serving as the axial ligand. Two single mutations were introduced, leading to the replacement of each methionine by a histidine residue. Characterization of both mutants, M78H and M84H cytochromes c555s, using biochemical and biophysical techniques has been carried out. The M84H mutant exhibits spectral features identical to those of native cytochrome. Its redox midpoint potential is decreased by 40 mV. By contrast, substitution of methionine 78 by a histidine residue strongly alters the structural and physicochemical properties of the molecule which exhibits characteristics of His/His iron coordination type rather than His/Met. These results allow us to identify methionine 78 as the sixth ligand of cytochrome c555s heme iron. Preliminary results on the thermostability of the native and mutant cytochromes c555 are also reported.

Cytochromes c555 from the hyperthermophilic bacterium Aquifex aeolicus (VF5). 1. Characterization of two highly homologous, soluble and membranous, cytochromes c555.

Two distinct class I (monoheme) c-type cytochromes from the hyperthermophilic bacterium Aquifex aeolicus were studied by biochemical and biophysical methods (i.e., optical and EPR spectroscopy, electrochemistry). The sequences of these two heme proteins (encoded by the cycB1 and cycB2 genes) are close to identical (85% identity in the common part of the protein) apart from the presence of an N-terminal stretch of 62 amino acid residues present only in the cycB1 gene. A soluble cytochrome was purified and identified by N-terminal sequencing as the cycB2 gene product. It showed an alpha-peak at 555 nm, an E(m) value of +220 mV, and electron paramagnetic resonance parameters of gz = 2.89, gy = 2.287, and gx = 1.52. A firmly membrane-bound cytochrome characterized by nearly identical properties was detected and attributed to the cycB1 gene product. The very high degree of homology of its N-terminal part to cytochrome c553 from Heliobacterium gestii strongly suggests it to be anchored to the membrane via N-terminally attached lipid molecules. The two heme proteins were named cytochrome c555s (soluble) and cytochrome c555m (membranous). Electron paramagnetic resonance on partially ordered membrane multilayers suggests that the solvent-exposed heme domain of cytochrome c555m is flexible with respect to the membrane plane. Possible functional roles for both cytochromes are discussed.

Bacterial origin for the isoprenoid biosynthesis enzyme HMG-CoA reductase of the archaeal orders Thermoplasmatales and Archaeoglobales.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase or HMGR) fulfills an essential role in archaea, as it is required for the synthesis of isoprenoid ethers, the main component of archaeal cell membranes. There are two clearly homologous but structurally different classes of the enzyme, one found mainly in eukaryotes and archaea (class 1), and the other found in bacteria (class 2). This feature facilitated the identification of several cases of interdomain lateral gene transfer (LGT), in particular, the bacterial origin for the HMGR gene from the archaeon Archaeoglobus fulgidus. In order to investigate if this LGT event was recent and limited in its scope or had a broad and long-term impact on the recipient and its related lineages, the HMGR gene was amplified and sequenced from a variety of archaea. The survey covered close relatives of A. fulgidus, the only archaeon known prior to this study to possess a bacterial-like HMGR; representatives of each main euryarchaeal group were also inspected. All culturable members of the archaeal group Archaeoglobales were found to display an HMGR very similar to the enzyme of the bacterium Pseudomonas mevalonii. Surprisingly, two species of the genus Thermoplasma also harbor an HMGR of bacterial origin highly similar to the enzymes found in the Archaeoglobales. Phylogenetic analyses of the HMGR gene and comparisons to reference phylogenies from other genes confirm a common bacterial origin for the HMGRs of Thermoplasmatales and Archaeoglobales. The most likely explanation of these results includes an initial bacteria-to-archaea transfer, followed by a another event between archaea. Their presence in two divergent archaeal lineages suggests an important adaptive role for these laterally transferred genes.

Phylogenetic analyses of two "archaeal" genes in thermotoga maritima reveal multiple transfers between archaea and bacteria.

The genome sequence of Thermotoga maritima revealed that 24% of its open reading frames (ORFs) showed the highest similarity scores to archaeal genes in BLAST analyses. Here we screened 16 strains from the genus Thermotoga and other related Thermotogales for the occurrence of two of these "archaeal" genes: the gene encoding the large subunit of glutamate synthase (gltB) and the myo-inositol 1P synthase gene (ino1). Both genes were restricted to the Thermotoga species within the Thermotogales. The distribution of the two genes, along with results from phylogenetic analyses, showed that they were acquired from Archaea during the divergence of the Thermotogales. Database searches revealed that three other bacteria-Dehalococcoides ethenogenes, Sinorhizobium meliloti, and Clostridium difficile-possess archaeal-type gltBs, and the phylogenetic analyses confirmed at least two lateral gene transfer (LGT) events between Bacteria and Archaea. These LGT events were also strongly supported by gene structure data, as the three domains in bacterial-type gltB are homologous to three independent ORFs in Archaea and Bacteria with archaeal-type gltBs. The ino1 gene has a scattered distribution among Bacteria, and apart from the Thermotoga strains it is found only in Aquifex aeolicus, D. ethenogenes, and some high-G+C Gram-positive bacteria. Phylogenetic analysis of the ino1 sequences revealed three highly supported prokaryotic clades, all containing a mixture of archaeal and bacterial sequences, and suggested that all bacterial ino1 genes had been recruited from archaeal donors. The Thermotoga strains and A. aeolicus acquired this gene independently from different archaeal species. Although transfer of genes from hyperthermophilic Archaea may have facilitated the evolution of bacterial hyperthermophily, between-domain transfers also affect mesophilic species. For hyperthermophiles, we hypothesize that LGT may be as much a consequence as the cause of adaptation to hyperthermophily.

Crystallization and preliminary X-ray analysis of adenylylsulfate reductase from Archaeoglobus fulgidus.

A group of anaerobic microorganisms use sulfate as the terminal electron acceptor for energy conservation. The process of sulfate reduction involves several enzymatic steps. One of them is the conversion of adenylyl sulfate (adenosine-5'-phosphosulfate) to sulfite, catalyzed by adenylylsulfate reductase. This enzyme is composed of a FAD-containing alpha-subunit and a beta-subunit harbouring two [4Fe-4S] clusters. Adenylylsulfate reductase was isolated from Archaeoglobus fulgidus under anaerobic conditions and crystallized using the hanging-drop vapour-diffusion method using PEG 4000 as precipitant. The crystals grew in space group P2(1)2(1)2(1), with unit-cell parameters a = 72.4, b = 113.2, c = 194.0 A. The asymmetric unit probably contains two alphabeta units. The crystals diffract beyond 2 A resolution and are suitable for X-ray structure analysis.

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.

Oxygen detoxification in the strict anaerobic archaeon Archaeoglobus fulgidus: superoxide scavenging by neelaredoxin.

Archaeoglobus fulgidus is a hyperthermophilic sulphate-reducing archaeon. It has an optimum growth temperature of 83 degrees C and is described as a strict anaerobe. Its genome lacks any homologue of canonical superoxide (O2.-) dismutases. In this work, we show that neelaredoxin (Nlr) is the main O2.- scavenger in A. fulgidus, by studying both the wild-type and recombinant proteins. Nlr is a 125-amino-acid blue-coloured protein containing a single iron atom/molecule, which in the oxidized state is high spin ferric. This iron centre has a reduction potential of +230 mV at pH 7.0. Nitroblue tetrazolium-stained gel assays of cell-soluble extracts show that Nlr is the main protein from A. fulgidus which is reactive towards O2.-. Furthermore, it is shown that Nlr is able to both reduce and dismutate O2.-, thus having a bifunctional reactivity towards O2.-. Kinetic and spectroscopic studies indicate that Nlr's superoxide reductase activity may allow the cell to eliminate O2.- quickly in a NAD(P)H-dependent pathway. On the other hand, Nlr's superoxide dismutation activity will allow the cell to detoxify O2.- independently of the cell redox status. Its superoxide dismutase activity was estimated to be 59 U mg-1 by the xanthine/xanthine oxidase assay at 25 degrees C. Pulse radiolysis studies with the isolated and reduced Nlr proved unambiguously that it has superoxide dismutase activity; at pH 7.1 and 83 degrees C, the rate constant is 5 x 106 M-1 s-1. Besides the superoxide dismutase activity, soluble cell extracts of A. fulgidus also exhibit catalase and NAD(P)H/oxygen oxidoreductase activities. By putting these findings together with the entire genomic data available, a possible oxygen detoxification mechanism in A. fulgidus is discussed.

Early evolution of cytochrome bc complexes.

Primary structures, functional characteristics and phylogenetic relationships of subunits of cytochrome bc complexes from phylogenetically diverse bacterial and archaeal species were analysed. A single case of lateral gene transfer, i.e. the import of an epsilon-proteobacterial cytochrome bc(1) complex into Aquificales, was identified. For the enzyme in the remainder of the species studied, the obtained phylogenies were globally in line with small subunit rRNA trees. The distribution of a few key phylogenetic markers, such as contiguousness of cytochrome b, nature of the c-type subunit or spacing between b-heme ligands, are discussed. A localised modification of previous tree topologies is proposed on the basis of the obtained data. The comparison of extant enzymes furthermore allowed us to define the minimal functional and evolutionary core of the enzyme. The data furthermore suggest that the ancestral enzyme was put together from subunits that previously had played a role in other electron transfer chains.

Adenylylsulfate reductases from archaea and bacteria are 1:1 alphabeta-heterodimeric iron-sulfur flavoenzymes--high similarity of molecular properties emphasizes their central role in sulfur metabolism.

Highly active adenylylsulfate (APS) reductase was isolated under N(2)/H(2) from sulfate-reducing and sulfide-oxidizing bacteria and archaea. It was a 1:1 alphabeta-heterodimer of molecular mass approximately 95 kDa, and two subunits (alpha approximately 75, beta approximately 20 kDa). The specific activity was 11-14 micromol (min mg)(-1); cofactor analysis revealed 0.96+/-0.05 FAD, 7.5+/-0.1 Fe and 7.9+/-0.25 S(2-). The photochemically reduced enzyme had a multiline EPR spectrum resulting from two interacting [4Fe-4S] centers. The properties of the different APS reductases were remarkably similar, although the enzyme is involved in different metabolic pathways and was isolated from phylogenetically far separated organisms. A structural model is proposed, with FAD bound to the alpha-subunit, and two [4Fe-4S] centers located in close proximity on the beta-subunit.

ATP synthesis at 100 degrees C by an ATPase purified from the hyperthermophilic archaeon Pyrodictium abyssi.

The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 lives close to 100 degrees C and gains energy by sulfur respiration, with hydrogen as electron donor. From the membranes of this hyperthermophile, an ATPase complex was isolated. The purified enzyme consists of six major polypeptides, the 67, 51, 41, 26 and 22 kDa subunits composing the AF(1) headpiece, and the 7 kDa proteolipid of the AF(0) component. The headpiece of the enzyme restored the formation of ATP during sulfur respiration in membrane vesicles from which it had been removed by low salt treatment. Characteristics of the reconstituted activity suggest that the same enzyme is responsible for ATP formation in untreated membranes. ATP formation was neither sensitive to ionophores and uncouplers, nor to dicyclohexyl carbodiimide, but depended on closed vesicles. Both ATPase activity (up to 2 micromol per min and mg protein) as well as ATP formation (up to 0.4 micromol per min and mg membrane protein) were highest at 100 degrees C. A P/e2 ratio of close to one can be estimated for sulfur respiration with hydrogen. In addition to ATP, autoradiographic detection revealed the formation of high quantities of (33)P(i)-labeled ADP and of another compound not identified so far.

Ignicoccus gen. nov., a novel genus of hyperthermophilic, chemolithoautotrophic Archaea, represented by two new species, Ignicoccus islandicus sp nov and Ignicoccus pacificus sp nov. and Ignicoccus pacificus sp. nov.

Two species of novel, chemolithoautotrophic, sulfidogenic micro-organisms were isolated from submarine hydrothermal systems in the Atlantic (at the Kolbeinsey Ridge north of Iceland) and in the Pacific (at 9 degrees N, 104 degrees W). The coccoid cells grew within a temperature range of 70-98 degrees C (optimum around 90 degrees C). They gained energy by reduction of elemental sulfur using molecular hydrogen as the electron donor. 165 rDNA-based sequence comparisons revealed that the organisms are members of the crenarchaeal branch of the Archaea. They represent a new, deeply branching lineage within the family of the Desulfurococcaceae. In DNA-DNA hybridization experiments both strains exhibited low levels of hybridization to each other and to further representatives of this family. Therefore, they represent a new genus, for which the name Ignicoccus gen. nov. is proposed. At present it consists of two new species, Ignicoccus islandicus sp. nov. (type strain is Kol8T = DSM 13165T = ATCC 700957T) and Ignicoccus pacificus sp. nov. (type strain is LPC33T = DSM 13166T = ATCC 700958T).

Direct proof for the presence and expression of two 5% different 16S rRNA genes in individual cells of Haloarcula marismortui.

A single-cell-derived pure culture of Haloarcula marismortui was obtained using the optical tweezers technique. Analysis of the 16S rRNA genes unambiguously demonstrated two 5% different 16S rRNA sequences. In addition, expression of both operons in the same cell was shown by fluorescence in situ hybridization with operon-specific probes.

Posttranscriptional modification of transfer RNA in the submarine hyperthermophile Pyrolobus fumarii.

In the RNA of hyperthermophiles, which grow optimally between 80 degrees C and 106 degrees C, posttranscriptional modification has been identified as a leading mechanism of structural stabilization. Particularly in the Archaeal evolutionary domain these modifications are expressed as a structurally diverse array of modification motifs, many of which include ribose methylation. Using mass spectrometric techniques we have examined the posttranscriptional modifications in unfractionated tRNA from the remarkable organism Pyrolobus fumarii, which grows optimally at 106 degrees C, but up to 113 degrees C (Blöchl et al. (1997), Extremophiles, 1, 14-21). Twenty-six modified nucleosides were detected, 11 of which are methylated in ribose. A new RNA nucleoside, 1,2'-O-dimethylguanosine (m1Gm) was characterized and the structure confirmed by chemical synthesis.

Carbon isotopic composition of individual Precambrian microfossils.

Ion microprobe measurements of carbon isotope ratios were made in 30 specimens representing six fossil genera of microorganisms petrified in stromatolitic chert from the approximately 850 Ma Bitter Springs Formation, Australia, and the approximately 2100 Ma Gunflint Formation, Canada. The delta 13C(PDB) values from individual microfossils of the Bitter Springs Formation ranged from -21.3 +/- 1.7% to -31.9 +/- 1.2% and the delta 13C(PDB) values from microfossils of the Gunflint Formation ranged from -32.4 +/- 0.7% to -45.4 +/- 1.2%. With the exception of two highly 13C-depleted Gunflint microfossils, the results generally yield values consistent with carbon fixation via either the Calvin cycle or the acetyl-CoA pathway. However, the isotopic results are not consistent with the degree of fractionation expected from either the 3-hydroxypropionate cycle or the reductive tricarboxylic acid cycle, suggesting that the microfossils studied did not use either of these pathways for carbon fixation. The morphologies of the microfossils suggest an affinity to the cyanobacteria, and our carbon isotopic data are consistent with this assignment.

Characterization of a novel lipid A containing D-galacturonic acid that replaces phosphate residues. The structure of the lipid a of the lipopolysaccharide from the hyperthermophilic bacterium Aquifex pyrophilus.

According to the 16 S rRNA phylogenetic tree, the hyperthermophilic bacterium Aquifex pyrophilus represents the deepest and shortest branching species of the kingdom Bacteria. We show for the first time that an organism, which is phylogenetically ancient on the basis of its 16 S rRNA and that exists at extreme conditions, may contain lipopolysaccharide (LPS). The LPS was extracted from dried bacteria using a modified phenol/water method. SDS-polyacrylamide gel electrophoresis and silver stain displayed a ladder-like pattern, which is typical for smooth-form LPS (possessing an O-specific polysaccharide). The molecular masses of the LPS populations were determined by matrix-assisted laser-desorption ionization mass spectrometry. Lipid A was precipitated after mild acid hydrolysis of LPS. Its complete structure was determined by chemical analyses, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser-desorption ionization mass spectrometry, and one- and two-dimensional NMR spectroscopy. The lipid A consists of a beta-(1-->6)-linked 2,3-diamino-2,3-dideoxy-D-glucopyranose (DAG) disaccharide carrying two residues each of (R)-3-hydroxytetradecanoic acid and (R)-3-hydroxyhexadecanoic acid in amide linkage and one residue of octadecanoic acid in ester linkage. Each DAG moiety carries one residue of each 3-hydroxytetradecanoic and 3-hydroxyhexadecanoic acid. In the nonreducing DAG, the octadecanoic acid is attached to the 3-hydroxy group of 3-hydroxytetradecanoic acid. Each DAG is substituted by one D-galacturonic acid residue, which is linked to O-1 of the reducing and to O-4 of the nonreducing end. This structure represents a novel type of lipid A.

The proteolipid of the A(1)A(0) ATP synthase from Methanococcus jannaschii has six predicted transmembrane helices but only two proton-translocating carboxyl groups.

The proteolipid, a hydrophobic ATPase subunit essential for ion translocation, was purified from membranes of Methanococcus jannaschii by chloroform/methanol extraction and gel chromatography and was studied using molecular and biochemical techniques. Its apparent molecular mass as determined in SDS-polyacrylamide gel electrophoresis varied considerably with the conditions applied. The N-terminal sequence analysis made it possible to define the open reading frame and revealed that the gene is a triplication of the gene present in bacteria. In some of the proteolipids, the N-terminal methionine is excised. Consequently, two forms with molecular masses of 21,316 and 21,183 Da were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The molecular and biochemical data gave clear evidence that the mature proteolipid from M. jannaschii is a triplication of the 8-kDa proteolipid present in bacterial F(1)F(0) ATPases and most archaeal A(1)A(0) ATPases. Moreover, the triplicated form lacks a proton-translocating carboxyl group in the first of three pairs of transmembrane helices. This finding puts in question the current view of the evolution of H(+) ATPases and has important mechanistic consequences for the structure and function of H(+) ATPases in general.

Construction of a highly bioluminescent Nitrosomonas as a probe for nitrification conditions.

Cloned luciferase-encoding operons were transferred by conjugation to a natural isolate of the ammonia-oxidizing bacterial strain Nitrosomonas sp. RST41-3, thereby establishing conjugation as a tool for gene transfer into Nitrosomonas strains. Luminescence was dependent on the pH of the medium and the concentration of the substrate ammonium chloride. Moreover, the luminescence of the transconjugants was reduced immediately by micromolar concentrations of nitrapyrin and allylthiourea, which are specific inhibitors of nitrification. Our results indicate that luminescent Nitrosomonas strains may be useful as a probe to detect nitrification conditions in the natural environment as well as in sewage plants.

Extremophiles and their adaptation to hot environments.

Water-containing terrestrial, subterranean and submarine high temperature areas harbor a variety of hyperthermophilic bacteria and archaea which are able to grow optimally above 80 degrees C. Hyperthermophiles are adapted to hot environments by their physiological and nutritional requirements. As a consequence, cell components like proteins, nucleic acids and membranes have to be stable and even function best at temperatures around 100 degrees C. The chemolithoautotrophic archaeon Pyrolobus fumarii is able to grow at 113 degrees C and, therefore, represents the upper temperature border of life. For the first time, (vegetative) cultures of Pyrolobus and Pyrodictium are able to survive autoclaving.