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1.  In vitro selection of an archaeal RNase P RNA mimics natural variation 
Archaea  2003;1(4):241-245.
Archaeal and bacterial RNase P RNAs are similar in sequence and secondary structure, but in the absence of protein, the archaeal RNAs are much less active and require extreme ionic conditions for activity. To assess how readily the activity of the archaeal RNA alone could be improved by small changes in sequence, in vitro selection was used to generate variants of a Methanobacterium formicicum RNase P RNA: Bacillus subtilus pre-tRNAAsp self-cleaving conjugate RNA. Functional variants were generated with a spectrum of mutations that were predominately consistent with natural variation in this RNA. Variants generated from the selection had cleavage rates comparable to that of wild type; variants with improved cleavage rates or lower ionic requirements were not obtained. This suggests that the RNase P RNAs of Bacteria and Archaea are globally optimized and the basis for the large biochemical differences between these two types of RNase P RNA is distributed in the molecule.
PMCID: PMC2685577  PMID: 15810433
Archaea; archaebacteria; ribonuclease P; ribozyme; SELEX
2.  Automated metabolic reconstruction for Methanococcus jannaschii  
Archaea  2003;1(4):223-229.
We present the computational prediction and synthesis of the metabolic pathways in Methanococcus jannaschii from its genomic sequence using the PathoLogic software. Metabolic reconstruction is based on a reference knowledge base of metabolic pathways and is performed with minimal manual intervention. We predict the existence of 609 metabolic reactions that are assembled in 113 metabolic pathways and an additional 17 super-pathways consisting of one or more component pathways. These assignments represent significantly improved enzyme and pathway predictions compared with previous metabolic reconstructions, and some key metabolic reactions, previously missing, have been identified. Our results, in the form of enzymatic assignments and metabolic pathway predictions, form a database (MJCyc) that is accessible over the World Wide Web for further dissemination among members of the scientific community.
PMCID: PMC2685575  PMID: 15810431
metabolic databases; Methanocaldococcus; pathway synthesis
3.  Minimal sulfur requirement for growth and sulfur-dependent metabolism of the hyperthermophilic archaeon Staphylothermus marinus  
Archaea  2003;1(3):191-197.
Staphylothermus marinus is an anaerobic hyperthermophilic archaeon that uses peptides as carbon and energy sources. Elemental sulfur (S°) is obligately required for its growth and is reduced to H2S. The metabolic functions and mechanisms of S° reduction were explored by examining S°-dependent growth and activities of key enzymes present in this organism. All three forms of S° tested—sublimed S°, colloidal S° and polysulfide—were used by S. marinus, and no other sulfur-containing compounds could replace S°. Elemental sulfur did not serve as physical support but appeared to function as an electron acceptor. The minimal S° concentration required for optimal growth was 0.05% (w/v). At this concentration, there appeared to be a metabolic transition from H2 production to S° reduction. Some enzymatic activities related to S°-dependent metabolism, including sulfur reductase, hydrogenase, glutamate dehydrogenase and electron transfer activities, were detected in cell-free extracts of S. marinus. These results indicate that S° plays an essential role in the heterotrophic metabolism of S. marinus. Reducing equivalents generated by the oxidation of amino acids from peptidolysis may be transferred to sulfur reductase and hydrogenase, which then catalyze the production of H2S and H2, respectively.
PMCID: PMC2685564  PMID: 15803665
electron transfer activity; glutamate dehydrogenase; hydrogen; hydrogenase; hyperthermophile; sulfide; sulfur reductase
4.  Calcium-induced aggregation of archaeal bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius  
Archaea  2003;1(3):175-183.
Previously, we showed that the proton permeability of small unilamellar vesicles (SUVs) composed of polar lipid fraction E (PLFE) from the thermoacidophilic archaeon Sulfolobus acidocaldarius was remarkably low and insensitive to temperature (Komatsu and Chong 1998). In this study, we used photon correlation spectroscopy to investigate the time dependence of PLFE SUV size as a function of Ca2+ concentration. In the absence of Ca2+, vesicle diameter changed little over 6 months. Addition of Ca2+, however, immediately induced formation of vesicle aggregates with an irregular shape, as revealed by confocal fluorescence microscopy. Aggregation was reversible upon addition of EDTA; however, the reversibility varied with temperature as well as incubation time with Ca2+. Freeze-fracture electron microscopy showed that, after a long period of incubation (2 weeks) with Ca2+, the PLFE vesicles had not just aggregated, but had fused or coalesced. The initial rate of vesicle aggregation varied sigmoidally with Ca2+ concentration. At pH 6.6, the threshold calcium concentration (Cr) for vesicle aggregation at 25 and 40 °C was 11 and 17 mM, respectively. At pH 3.0, the Cr at 25 °C increased to 25 mM. The temperature dependence of Cr may be attributable to changes in membrane surface potential, which was –22.0 and –13.2 mV at 25 and 40 °C, respectively, at pH 6.6, as determined by 2-(p-toluidinyl)naphthalene-6-sulfonic acid fluorescence. The variation in surface potential with temperature is discussed in terms of changes in lipid conformation and membrane organization.
PMCID: PMC2685566  PMID: 15803663
fluorescence; light scattering; membranes; microscopy; pH; surface potential; temperature; vesicle size
5.  Remarkable sequence signatures in archaeal genomes 
Archaea  2003;1(3):185-190.
Complete archaeal genomes were probed for the presence of long (≥ 25 bp) oligonucleotide repeats (words). We detected the presence of many words distributed in tandem with narrow ranges of periodicity (i.e., spacer length between repeats). Similar words were not identified in genomes of non-archaeal species, namely Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Mycoplasma genitalium and Mycoplasma pneumoniae. BLAST similarity searches against the GenBank nucleotide sequence database revealed that these words were archaeal species-specific, indicating that they are of a signature character. Sequence analysis and genome viewing tools showed these repeats to be restricted to non-coding regions. Thus, archaea appear to possess a non-coding genomic signature that is absent in bacterial species. The identification of a species-specific genomic signature would be of great value to archaeal genome mapping, evolutionary studies and analyses of genome complexity.
PMCID: PMC2685567  PMID: 15803664
Archaea; bioinformatics; comparative genomics; genome signature; oligonucleotide frequencies
6.  Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen 
Archaea  2003;1(3):151-164.
Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine-labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N-ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N-ethylmaleimide failed to inhibit uptake of the four other rhodamine-labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis ofarchaeosomes rich in surface-exposed phosphoserine head groups via a phosphatidylserine receptor, and energy-independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL-12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation.
PMCID: PMC2685569  PMID: 15803661
antibody; archaea; cytotoxic T lymphocyte; liposomes; phagocytosis; phosphatidylserine

Results 1-6 (6)