Pyrococcus furiosus laminarinase (LamA, PF0076) is an endo-glycosidase that hydrolyzes β-1,3-gluco-oligosaccharides, but not β-1,4-gluco-oligosaccharides. We studied the specificity of LamA towards small saccharides by using 4-methylumbelliferyl β-glucosides with different linkages. Besides endo-activity, wild-type LamA has some exo-activity, and catalyzes the hydrolysis of mixed-linked oligosaccharides (Glcβ4Glcβ3Glcβ-MU (Glc = glucosyl, MU = 4-methylumbelliferyl)) with both β-1,4 and β-1,3 specificities. The LamA mutant E170A had severely reduced hydrolytic activity, which is consistent with Glu170 being the catalytic nucleophile. The E170A mutant was active as a glycosynthase, catalyzing the condensation of α-laminaribiosyl fluoride to different acceptors. The best condensation yields were found at pH 6.5 and 50 °C, but did not exceed 30%. Depending on the acceptor, the synthase generated either a β-1,3 or a β-1,4 linkage.

J. Van Lieshout

M. Faijes

J. Nieto

J. Van Der Oost

A. Planas

Hydrolase and glycosynthase activity of endo-1,3-β-glucanase from the thermophile Pyrococcus furiosus

Pyrococcus species are hyperthermophilic members of the order Thermococcales, with optimal growth temperatures approaching 100 °C. All species grow heterotrophically and produce H2 or, in the presence of elemental sulfur (S°), H2S. Pyrococcus woesei and P. furiosus were isolated from marine sediments at the same Vulcano Island beach site and share many morphological and physiological characteristics. We report here that the rDNA operons of these strains have identical sequences, including their intergenic spacer regions and part of the 23S rRNA. Both species grow rapidly and produce H2 in the presence of 0.1% maltose and 10–100 µM sodium tungstate in S°-free medium. However,P. woesei shows more extensive autolysis than P. furiosus in the stationary phase. Pyrococcusfuriosus and P. woesei share three closely related families of insertion sequences (ISs). A Southern blot performed with IS probes showed extensive colinearity between the genomes of P. woesei and P. furiosus. Cloning and sequencing of ISs that were in different contexts in P. woesei and P. furiosus revealed that the napA gene in P. woesei is disrupted by a type III IS element, whereas in P. furiosus, this gene is intact. A type I IS element, closely linked to the napA gene, was observed in the same context in both P. furiosus and P. woesei genomes. Our results suggest that the IS elements are implicated in genomic rearrangements and reshuffling in these closely related strains. We propose to rename P. woesei a subspecies of P. furiosus based on their identical rDNA operon sequences, many common IS elements that are shared genomic markers, and the observation that all P. woesei nucleotide sequences deposited in GenBank to date are &gt; 99% identical to P. furiosus sequences.

Wirojne Kanoksilapatham

Juan M. González

Dennis L. Maeder

Jocelyne DiRuggiero

Frank T. Robb

A proposal to rename the hyperthermophile Pyrococcus woesei as Pyrococcus furiosus subsp. woesei

The signal recognition particle (SRP) RNA helix 6 of archaea and eukaryotes is essential for the binding of protein SRP19 and the assembly of a functional complex. The conserved adenosine at the third position of the tetraloop of helix 6 (A149) is crucial for the binding of protein SRP19 in the mammalian SRP. Here we investigated the significance of the equivalent adenosine residue at position 159 (A159) of Archaeoglobus fulgidus SRP RNA. The A159 of A. fulgidus and A149 of human SRP RNA were changed to C, G or U, and fragments containing helix 6 or helices 6 and 8 were synthesized by run-off transcription with T7 RNA polymerase. The ability of recombinant A. fulgidus and human SRP19 to form ribonucleoprotein complexes was measured in vitro. The simultaneous presence of A149 and helix 8 is required for the high-affinity binding of SRP19 to the human SRP RNA. In contrast, A. fulgidus SRP19 binds to the SRP RNA fragments with high affinity irrespective of the nature of the nucleotide, demonstrating that A159 does not directly participate in protein binding. Instead, as indicated by the resistance of the wild-type A. fulgidus RNA towards digestion by RNase A, this residue allows the formation of a tightly folded RNA molecule. The high affinity between A. fulgidus SRP19 and RNA molecules that contain both helices 6 and 8 suggests that A159 is likely to initiate archaeal SRP assembly by forming a conserved tertiary RNA– RNA interaction.

Jiaming Yin

Qiaojia Huang

Olga  N. Pakhomova

Andrew  P. Hinck

Christian Zwieb

The conserved adenosine in helix 6 of Archaeoglobus fulgidus signal recognition particle RNA initiates SRP assembly

A hyperthermophilic archaeal strain, KOD1, isolated from a solfatara on Kodakara Island, Japan, has previously been reported as Pyrococcus sp. KOD1. However, a detailed phylogenetic tree, made possible by the recent accumulation of 16S rRNA sequences of various species in the order Thermococcales, indicated that strain KOD1 is a member of the genus Thermococcus. We performed DNA&#8211;DNA hybridization tests against species that displayed high similarity in terms of 16S ribosomal DNA sequences, including Thermococcus peptonophilus and Thermococcus stetteri. Hybridization results and differences in growth characteristics and substrate utilization differentiated strain KOD1 from T. peptonophilus and T. stetteri at the species level. Our results indicate that strain KOD1 represents a new species of Thermococcus, which we designate as Thermococcus kodakaraensis KOD1 sp. nov.

Haruyuki Atomi

Toshiaki Fukui

Tamotsu Kanai

Masaaki Morikawa

Tadayuki Imanaka

Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1

The Methanocaldococcus jannaschii genome contains putative genes for all four nonoxidative pentose phosphate pathway enzymes. Open reading frame (ORF) MJ0960 is a member of the mipB/talC family of ‘transaldolase-like’ genes, so named because of their similarity to the well-characterized transaldolase B gene family. However, recently, it has been reported that both the mipB and the talC genes from Escherichia coli encode novel enzymes with fructose-6-phosphate aldolase activity, not transaldolase activity (Schürmann and Sprenger 2001). The same study reports that other members of the mipB/talC family appear to encode transaldolases. To confirm the function of MJ0960 and to clarify the presence of a nonoxidative pentose phosphate pathway in M. jannaschii, we have cloned ORF MJ0960 from M. jannaschii genomic DNA and purified the recombinant protein. MJ0960 encodes a transaldolase and displays no fructose-6-phosphate aldolase activity. It retained full activity for 4 h at 80 °C, and for 3 weeks at 25 °C. Methanocaldococcus jannaschii transaldolase has a maximal velocity (Vmax) of 1.0 ± 0.2 µmol min–1 mg–1 at 25 °C, whereas Vmax = 12.0 ± 0.5 µmol min–1 mg–1 at 50 °C. Apparent Michaelis constants at 50 °C were Km = 0.65 ± 0.09 mM for fructose-6-phosphate and Km = 27.8 ± 4.3 µM for erythrose-4-phosphate. When ribose-5-phosphate replaced erythrose-4-phosphate as an aldose acceptor, Vmax decreased twofold, whereas the Km was 150-fold higher. The molecular mass of the active enzyme is 271 ± 27 kDa as estimated by gel filtration, whereas the predicted monomer size is 23.96 kDa, suggesting that the native form of the protein is probably a decamer. A readily available source of thermophilic pentose phosphate pathway enzymes including transaldolase may have direct application in enzymatic biohydrogen production.

Tim Soderberg

Robert C. Alver

Transaldolase of Methanocaldococcus jannaschii

A yeast two-hybrid system was used to identify protein–protein interactions between the ribonuclease P (RNase P) protein subunits Mth11p, Mth687p, Mth688p and Mth1618p from the archaeon Methanothermobacter thermoautotrophicus. Clear interactions between Mth688p and Mth687p, and between Mth1618p and Mth11p, were confirmed by HIS3 and LacZ reporter expression. Weaker interactions of Mth687p and Mth688p with Mth11p, and Mth11p with itself, are also suggested. These interactions resemble, and confirm, those previously seen among the homologs of these proteins in the more complex yeast RNase P holoenzyme.

Thomas A. Hall

James  W. Brown

Interactions between RNase P protein subunits in Archaea

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.

Daniel Williams

James W. Brown

In vitro selection of an archaeal RNase P RNA mimics natural variation

All of the known self-transmissable plasmids of the Archaea have been found in the genus Sulfolobus. To gain more insight into archaeal conjugative processes, four newly isolated self-transmissable plasmids, pKEF9, pHVE14, pARN3 and pARN4, were sequenced and subjected to a comparative sequence analysis with two earlier sequenced plasmids, pNOB8 and pING1. The analyses revealed three conserved and functionally distinct sections in the genomes. Section A is considered to encode the main components of the conjugative apparatus, where two genes show low but significant sequence similarity to sections of genes encoding bacterial conjugative proteins. A putative origin of replication is located in section B, which is highly conserved in sequence and contains several perfect and imperfect direct and inverted repeats. Further downstream, in section C, an operon encoding six to nine smaller proteins is implicated in the initiation and regulation of replication. Each plasmid carries an integrase gene of the type that does not partition on integration, and there is strong evidence for their integration into host chromosomes, where they may facilitate intercellular exchange of chromosomal genes. Two plasmids contain hexameric short regularly spaced repeats (SRSR), which have been implicated in plasmid maintenance, and each plasmid carries multiple recombination motifs, concentrated in the variable regions, which likely provide sites for genomic rearrangements.

Bo Greve

Susanne Jensen

Kim Brügger

Wolfram Zillig

Roger A. Garrett

Genomic comparison of archaeal conjugative plasmids from Sulfolobus

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.

Sophia Tsoka

David Simon

Christos A. Ouzounis

Automated metabolic reconstruction for Methanococcus jannaschii

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Archaea publishes original research articles as well as review articles dealing with all aspects of archaea, providing a unique venue for exchanging information about these extraordinary prokaryotes.

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