The acyl-adenylate-forming enzyme superfamily, consisting of acyl- and aryl-CoA synthetases, the adenylation domain of the nonribosomal peptide synthetases, and luciferase, has three signature motifs (I–III) and ten conserved core motifs (A1–A10), some of which overlap the signature motifs. The consensus sequence for signature motif III (core motif A7) in acetyl-CoA synthetase is Y-X-S/T/A-G-D, with an invariant fifth position, highly conserved first and fourth positions, and variable second and third positions. Kinetic studies of enzyme variants revealed that an alteration at any position resulted in a strong decrease in the catalytic rate, although the most deleterious effects were observed when the first or fifth positions were changed. Structural modeling suggests that the highly conserved Tyr in the first position plays a key role in active site architecture through interaction with a highly conserved active-site Gln, and the invariant Asp in the fifth position plays a critical role in ATP binding and catalysis through interaction with the 2′- and 3′-OH groups of the ribose moiety. Interactions between these Asp and ATP are observed in all structures available for members of the superfamily, consistent with a critical role in substrate binding and catalysis for this invariant residue.

Methanosarcina mazei is one of the model organisms for the methanogenic order Methanosarcinales whose metabolism has been studied in detail. However, the genetic toolbox is still limited. This study was aimed at widening the scope of utilizable methods in this group of organisms. (i) Proteins specific to methanogens are oftentimes difficult to produce in E. coli. However, a protein production system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins in Ms. mazei. The promoter p1687, which directs the transcription of methyl transferases that demethylate methylamines, was cloned into plasmid pWM321 and its activity was determined by monitoring β-glucuronidase production. The promoter was inactive during growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding the β-glucuronidase from E. coli was fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was overproduced in Ms. mazei and was purified in an active form by affinity chromatography. (ii) Puromycin is currently the only antibiotic used as a selectable marker in Ms. mazei and its relatives. We established neomycin resistance as a second selectable marker by designing a plasmid that confers neomycin resistance in Ms. mazei.

The influence of pH (6.0; 7.0; 8.0) of the growth medium of Aeropyrum pernix K1 on the structural organization and fluidity of archaeosomes prepared from a polar-lipid methanol fraction (PLMF) was investigated using fluorescence anisotropy and electron paramagnetic resonance (EPR) spectroscopy. Fluorescence anisotropy of the lipophilic fluorofore 1,6-diphenyl-1,3,5-hexatriene and empirical correlation time of the spin probe methylester of 5-doxylpalmitate revealed gradual changes with increasing temperature for the pH. A similar effect has been observed by using the trimethylammonium-6-diphenyl-1,3,5-hexatriene, although the temperature changes were much smaller. As the fluorescence steady-state anisotropy and the empirical correlation time obtained directly from the EPR spectra alone did not provide detailed structural information, the EPR spectra were analysed by computer simulation. This analysis showed that the archaeosome membranes are heterogeneous and composed of several regions with different modes of spin-probe motion at temperatures below 70°C. At higher temperatures, these membranes become more homogeneous and can be described by only one spectral component. Both methods indicate that the pH of the growth medium of A. pernix does not significantly influence its average membrane fluidity. These results are in accordance with TLC analysis of isolated lipids, which show no significant differences between PLMF isolated from A. pernix grown in medium with different pH.

Many archaeal cell envelopes contain a protein coat or sheath composed of one or more surface exposed proteins. These surface layer (S-layer) proteins contribute structural integrity and protect the lipid membrane from environmental challenges. To explore the species diversity of these layers in the Methanosarcinaceae, the major S-layer protein in Methanosarcina barkeri strain Fusaro was identified using proteomics. The Mbar_A1758 gene product was present in multiple forms with apparent sizes of 130, 120, and 100 kDa, consistent with post-translational modifications including signal peptide excision and protein glycosylation. A protein with features related to the surface layer proteins found in Methanosarcina acetivorans C2A and Methanosarcina mazei Goel was identified in the M. barkeri genome. These data reveal a distinct conserved protein signature with features and implied cell surface architecture in the Methanosarcinaceae that is absent in other archaea. Paralogous gene expression patterns in two Methanosarcina species revealed abundant expression of a single S-layer paralog in each strain. Respective promoter elements were identified and shown to be conserved in mRNA coding and upstream untranslated regions. Prior M. acetivorans genome annotations assigned S-layer or surface layer associated roles of eighty genes: however, of 68 examined none was significantly expressed relative to the experimentally determined S-layer gene.

Novel cardiolipins from Archaea were detected by screening the intact polar lipid (IPL) composition of microbial communities associated with methane seepage in deep-sea sediments from the Pakistan margin by high-performance liquid chromatography electrospray ionization mass spectrometry. A series of tentatively identified cardiolipin analogues (dimeric phospholipids or bisphosphatidylglycerol, BPG) represented 0.5% to 5% of total archaeal IPLs. These molecules are similar to the recently described cardiolipin analogues with four phytanyl chains from extreme halophilic archaea. It is worth noting that cardiolipin analogues from the seep archaeal communities are composed of four isoprenoidal chains, which may contain differences in chain length (20 and 25 carbon atoms) and degrees of unsaturation and the presence of a hydroxyl group. Two novel diether lipids, structurally related to the BPGs, are described and interpreted as degradation products of archaeal cardiolipin analogues. Since archaeal communities in seep sediments are dominated by anaerobic methanotrophs, our observations have implications for characterizing structural components of archaeal membranes, in which BPGs are presumed to contribute to modulation of cell permeability properties. Whether BPGs facilitate interspecies interaction in syntrophic methanotrophic consortia remains to be tested.

A part of the biosynthetic pathway of archaeal membrane lipids, comprised of 4 archaeal enzymes, was reconstructed in the cells of Escherichia coli. The genes of the enzymes were cloned from a mesophilic methanogen, Methanosarcina acetivorans, and the activity of each enzyme was confirmed using recombinant proteins. In vitro radioassay showed that the 4 enzymes are sufficient to synthesize an intermediate of archaeal membrane lipid biosynthesis, that is, 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate, from precursors that can be produced endogenously in E. coli. Introduction of the 4 genes into E. coli resulted in the production of archaeal-type lipids. Detailed liquid chromatography/electron spray ionization-mass spectrometry analyses showed that they are metabolites from the expected intermediate, that is, 2,3-di-O-geranylgeranyl-sn-glycerol and 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphoglycerol. The metabolic processes, that is, dephosphorylation and glycerol modification, are likely catalyzed by endogenous enzymes of E. coli.

The availability of in vitro assembly systems to produce recombinant archaeal RNA polymerases (RNAPs) offers one of the most powerful experimental tools for investigating the still relatively poorly understood molecular mechanisms underlying RNAP function. Over the last few years, we pioneered new robot-based high-throughput mutagenesis approaches to study structure/function relationships within various domains surrounding the catalytic center. The Bridge Helix domain, which appears in numerous X-ray structures as a 35-amino-acid-long alpha helix, coordinates the concerted movement of several other domains during catalysis through kinking of two discrete molecular hinges. Mutations affecting these kinking mechanisms have a direct effect on the specific catalytic activity of RNAP and can in some instances more than double it. Molecular dynamics simulations have established themselves as exceptionally useful for providing additional insights and detailed models to explain the underlying structural motions.

The genome sequence of Haloferax volcanii is available and several comparative genomic in silico studies were performed that yielded novel insight for example into protein export, RNA modifications, small non-coding RNAs, and ubiquitin-like Small Archaeal Modifier Proteins. The full range of functional genomic methods has been established and results from transcriptomic, proteomic and metabolomic studies are discussed. Notably, Hfx. volcanii is together with Halobacterium salinarum the only prokaryotic species for which a translatome analysis has been performed. The results revealed that the fraction of translationally-regulated genes in haloarchaea is as high as in eukaryotes. A highly efficient genetic system has been established that enables the application of libraries as well as the parallel generation of genomic deletion mutants. Facile mutant generation is complemented by the possibility to culture Hfx. volcanii in microtiter plates, allowing the phenotyping of mutant collections. Genetic approaches are currently used to study diverse biological questions–from replication to posttranslational modification—and selected results are discussed. Taken together, the wealth of functional genomic and genetic tools make Hfx. volcanii a bona fide archaeal model species, which has enabled the generation of important results in recent years and will most likely generate further breakthroughs in the future.

Prolidases hydrolyze Xaa-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus (OP) compounds, including the nerve agents soman and sarin. Ph1prol (PH0974) has previously been isolated and characterized from Pyrococcus horikoshii and was shown to have higher catalytic activity over a broader pH range, higher affinity for metal, and increased thermostability compared to P. furiosus prolidase, Pfprol (PF1343). To obtain a better enzyme for OP nerve agent decontamination and to investigate the structural factors that may influence protein thermostability and thermoactivity, randomly mutated Ph1prol enzymes were prepared. Four Ph1prol mutants (A195T/G306S-, Y301C/K342N-, E127G/E252D-, and E36V-Ph1prol) were isolated which had greater thermostability and improved activity over a broader range of temperatures against Xaa-Pro dipeptides and OP nerve agents compared to wild type Pyrococcus prolidases.

RNase P is a highly conserved ribonucleoprotein enzyme that represents a model complex for understanding macromolecular RNA-protein interactions. Archaeal RNase P consists of one RNA and up to five proteins (Pop5, RPP30, RPP21, RPP29, and RPP38/L7Ae). Four of these proteins function in pairs (Pop5-RPP30 and RPP21–RPP29). We have used nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to characterize the interaction between Pop5 and RPP30 from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu). NMR backbone resonance assignments of free RPP30 (25 kDa) indicate that the protein is well structured in solution, with a secondary structure matching that observed in a closely related crystal structure. Chemical shift perturbations upon the addition of Pop5 (14 kDa) reveal its binding surface on RPP30. ITC experiments confirm a net 1 : 1 stoichiometry for this tight protein-protein interaction and exhibit complex isotherms, indicative of higher-order binding. Indeed, light scattering and size exclusion chromatography data reveal the complex to exist as a 78 kDa heterotetramer with two copies each of Pop5 and RPP30. These results will inform future efforts to elucidate the functional role of the Pop5-RPP30 complex in RNase P assembly and catalysis.

It is becoming clear that the regulation of gas vesicle biogenesis in Halobacterium salinarum NRC-1 is multifaceted and appears to integrate environmental and metabolic cues at both the transcriptional and posttranscriptional levels. The mechanistic details underlying this process, however, remain unclear. In this manuscript, we quantify the contribution of light scattering made by both intracellular and released gas vesicles isolated from Halobacterium salinarum NRC-1, demonstrating that each form can lead to distinct features in growth curves determined by optical density measured at 600 nm (OD600). In the course of the study, we also demonstrate the sensitivity of gas vesicle accumulation in Halobacterium salinarum NRC-1 on small differences in growth conditions and reevaluate published works in the context of our results to present a hypothesis regarding the roles of the general transcription factor tbpD and the TCA cycle enzyme aconitase on the regulation of gas vesicle biogenesis.

Extreme environments, generally characterized by atypical temperatures, pH, pressure, salinity, toxicity, and radiation levels, are inhabited by various microorganisms specifically adapted to these particular conditions, called extremophiles. Among these, the microorganisms belonging to the Archaea domain are of significant biotechnological importance as their biopolymers possess unique properties that offer insights into their biology and evolution. Particular attention has been devoted to two main types of biopolymers produced by such peculiar microorganisms, that is, the extracellular polysaccharides (EPSs), considered as a protection against desiccation and predation, and the endocellular polyhydroxyalkanoates (PHAs) that provide an internal reserve of carbon and energy. Here, we report the composition, biosynthesis, and production of EPSs and PHAs by different archaeal species.

Disulfide bonds are generally not used to stabilize proteins in the cytosolic compartments of bacteria or eukaryotic cells, owing to the chemically reducing nature of those environments. In contrast, certain thermophilic archaea use disulfide bonding as a major mechanism for protein stabilization. Here, we provide a current survey of completely sequenced genomes, applying computational methods to estimate the use of disulfide bonding across the Archaea. Microbes belonging to the Crenarchaeal branch, which are essentially all hyperthermophilic, are universally rich in disulfide bonding while lesser degrees of disulfide bonding are found among the thermophilic Euryarchaea, excluding those that are methanogenic. The results help clarify which parts of the archaeal lineage are likely to yield more examples and additional specific data on protein disulfide bonding, as increasing genomic sequencing efforts are brought to bear.

A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP). The nonreplicating allelic exchange vector (pRS345) carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA154. Characterizing M. mazeiΔsRNA154 under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154 in regulation of nitrogen fixation by posttranscriptional regulation.

Picrophilus oshimae and Picrophilus torridus are free-living, moderately thermophilic and acidophilic organisms from the lineage of Euryarchaeota. With a pH optimum of growth at pH 0.7 and the ability to even withstand molar concentrations of sulphuric acid, these organisms represent the most extreme acidophiles known. So far, nothing is known about plasmid biology in these hyperacidophiles. Also, there are no genetic tools available for this genus. We have mobilized the 7.6 Kbp plasmid from P. oshimae in E. coli by introducing origin-containing transposons and described the plasmid in terms of its nucleotide sequence, copy number in the native host, mode of replication, and transcriptional start sites of the encoded ORFs. Plasmid pPO1 may encode a restriction/modification system in addition to its replication functions. The information gained from the pPO1 plasmid may prove useful in developing a cloning system for this group of extreme acidophiles.

In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096) were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays. The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR. The phenotypic analyses of the disruptants confirmed that ORF Saci_1227 encodes a DNA photolyase which functions in vivo, but they could not implicate ORF Saci_1096 in repair of UV- or other externally induced DNA damage despite its similarity to genes encoding UV damage endonucleases. The success of the gene-disruption strategy, which used 5′ extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains.

The hydrogenotrophic methanogens Methanothermobacter marburgensis and Methanothermobacter thermautotrophicus can easily be mass cultured. They have therefore been used almost exclusively to study the biochemistry of methanogenesis from H2 and CO2, and the genomes of these two model organisms have been sequenced. The close relationship of the two organisms is reflected in their genomic architecture and coding potential. Within the 1,607 protein coding sequences (CDS) in common, we identified approximately 200 CDS required for the synthesis of the enzymes, coenzymes, and prosthetic groups involved in CO2 reduction to methane and in coupling this process with the phosphorylation of ADP. Approximately 20 additional genes, such as those for the biosynthesis of F430 and methanofuran and for the posttranslational modifications of the two methyl-coenzyme M reductases, remain to be identified.

DNA microarray technology allows for a quick and easy comparison of complete transcriptomes, resulting in improved molecular insight in fluctuations of gene expression. After emergence of the microarray technology about a decade ago, the technique has now matured and has become routine in many molecular biology laboratories. Numerous studies have been performed that have provided global transcription patterns of many organisms under a wide range of conditions. Initially, implementation of this high-throughput technology has lead to high expectations for ground breaking discoveries. Here an evaluation is performed of the insight that transcriptome analysis has brought about in the field of hyperthermophilic archaea. The examples that will be discussed have been selected on the basis of their impact, in terms of either biological insight or technological progress.

Methanogens are the only known microorganisms capable of methane production, making them of interest when investigating methane abatement strategies. A number of experiments have been conducted to study the methanogen population in the rumen of cattle and sheep, as well as the relationship that methanogens have with other microorganisms. The rumen methanogen species differ depending on diet and geographical location of the host, as does methanogenesis, which can be reduced by modifying dietary composition, or by supplementation of monensin, lipids, organic acids, or plant compounds within the diet. Other methane abatement strategies that have been investigated are defaunation and vaccines. These mitigation methods target the methanogen population of the rumen directly or indirectly, resulting in varying degrees of efficacy. This paper describes the methanogens identified in the rumens of cattle and sheep, as well as a number of methane mitigation strategies that have been effective in vivo.

The human body (primarily the intestinal tract, the oral cavity, and the skin) harbours approximately 1,000 different bacterial species. However, the number of archaeal species known to colonize man seems to be confined to a handful of organisms within the class Euryarchaeota (including Methanobrevibacter smithii, M. oralis, and Methanosphaera stadtmanae). In contrast to this conspicuously low diversity of Archaea in humans their unique physiology in conjunction with the growing number of reports regarding their occurrence at sites of infection has made this issue an emerging field of study. While previous review articles in recent years have addressed the putative role of particularly methanogenic archaea for human health and disease, this paper compiles novel experimental data that have been reported since then. The aim of this paper is to inspire the scientific community of “Archaea experts” for those unique archaeal organisms that have successfully participated in the human-microbe coevolution.

With the availability of a genome sequence and increasingly sophisticated genetic tools, Haloferax volcanii is becoming a model for both Archaea and halophiles. In order for H. volcanii to reach a status equivalent to Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22 H. volcanii deletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of an H. volcanii mutant library and suggest that they should form the basis of a larger genome-wide experiment.

Methanohalophilus mahii is the type species of the genus Methanohalophilus, which currently comprises three distinct species with validly published names. Mhp. mahii represents moderately halophilic methanogenic archaea with a strictly methylotrophic metabolism. The type strain SLPT was isolated from hypersaline sediments collected from the southern arm of Great Salt Lake, Utah. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,012,424 bp genome is a single replicon with 2032 protein-coding and 63 RNA genes and part of the Genomic Encyclopedia of Bacteria and Archaea project. A comparison of the reconstructed energy metabolism in the halophilic species Mhp. mahii with other representatives of the Methanosarcinaceae reveals some interesting differences to freshwater species.

Heme is an essential prosthetic group for many proteins involved in fundamental biological processes in all three domains of life. In Eukaryota and Bacteria heme is formed via a conserved and well-studied biosynthetic pathway. Surprisingly, in Archaea heme biosynthesis proceeds via an alternative route which is poorly understood. In order to formulate a working hypothesis for this novel pathway, we searched 59 completely sequenced archaeal genomes for the presence of gene clusters consisting of established heme biosynthetic genes and colocalized conserved candidate genes. Within the majority of archaeal genomes it was possible to identify such heme biosynthesis gene clusters. From this analysis we have been able to identify several novel heme biosynthesis genes that are restricted to archaea. Intriguingly, several of the encoded proteins display similarity to enzymes involved in heme d1 biosynthesis. To initiate an experimental verification of our proposals two Methanosarcina barkeri proteins predicted to catalyze the initial steps of archaeal heme biosynthesis were recombinantly produced, purified, and their predicted enzymatic functions verified.

The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea, separating the two strands of chromosomal DNA during replication. The catalytic activity resides within the C-terminal region of the MCM protein, while the N-terminal portion plays an important role in DNA binding and protein multimerization. An alignment of MCM homologues from several archaeal species revealed a number of conserved amino acids. Here several of the conserved residues located on the surface of the helicase have been mutated and their roles in MCM functions determined. It was found that some mutations result in increased affinity for ssDNA while the affinity for dsDNA is decreased. Other mutants exhibit the opposite effect. Thus, the data suggest that these conserved surface residues may participate in MCM-DNA interactions.