Pernix K1 Research Articles

Microbial genomics

The 1.6-Mb sequence of the genome of the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1 has been published by staff at the National Institute of Technology and Evaluation, Tokyo, Japan 1 Kawarabayasi, Y. et al. (1999) Complete genome sequence of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1, DNA Res. 6, 83–101, 145–152 Google Scholar . A. pernix was first isolated from a coastal solfataric thermal vent on Kodakara-Jima Island, Japan in 1993, grows optimally at 95°C and was the first strictly aerobic hyperthermophilic archaeon to be described. Of the 2694 open reading frames (ORFs) identified in the genome sequence, over half do not show any significant similarity to known sequences. All the enzymes of the tricarboxylic-acid cycle are encoded in the genome, except for α-ketoglutarate dehydrogenase (which might be functionally replaced by 2-oxoacid:ferredoxin oxidoreductase). Intragenomic sequence comparisons suggest that a considerable number of ORFs were generated by sequence duplication. Fourteen of the 50 RNA genes contain introns. An online genome database for A. pernix is available on http://www.mild.nite.go.jp/. The genome sequence is also available in seven segments from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html)

Purification and characterization of an intracellular heat-stable proteinase (pernilase) from the marine hyperthermophilic archaeon Aeropyrum pernix K1.

A novel intracellular serine proteinase from the marine aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) that we designated pernilase was purified by ammonium sulfate precipitation, anionic-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 50 kDa as determined by SDS-PAGE. The proteinase had a broad pH profile (pH 5-10) with an optimum pH of 9.0 for peptide hydrolysis. The optimum temperature for enzyme activity was 90 degrees C. The enzyme was strongly inhibited by diisopropyl fluorophosphate (DFP) and phenylmethyl sulfonylfluoride (PMSF), suggesting that it corresponds to a serine proteinase. The enzyme was highly resistant to the reducing agents dithiothreitol and 2-mercaptoethanol but sensitive to the denaturing reagents guanidine-HCl and urea and also to the detergent sodium dodecyl sulfate (SDS). Pernilase showed high substrate specificity for Boc-Leu-Gly-Arg-MCA peptide. Thermostability of this enzyme showed half-lives of 85min at 100 degrees C and 12 min at 110 degrees C.

Complete genome sequence of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1.

The complete sequence of the genome of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1, which optimally grows at 95 degrees C, has been determined by the whole genome shotgun method with some modifications. The entire length of the genome was 1,669,695 bp. The authenticity of the entire sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2,694 open reading frames (ORFs) were assigned. By similarity search against public databases, 633 (23.5%) of the ORFs were related to genes with putative function and 523 (19.4%) to the sequences registered but with unknown function. All the genes in the TCA cycle except for that of alpha-ketoglutarate dehydrogenase were included, and instead of the alpha-ketoglutarate dehydrogenase gene, the genes coding for the two subunits of 2-oxoacid:ferredoxin oxidoreductase were identified. The remaining 1,538 ORFs (57.1%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs suggested that a considerable member of ORFs were generated by sequence duplication. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 47 tRNA genes including 14 genes with intron structures. All the assigned ORFs and RNA coding regions occupied 89.12% of the whole genome. The data presented in this paper are available on the internet homepage (http://www.mild.nite.go.jp).

A novel phosphoglycolipid archaetidyl(glucosyl)inositol with two sesterterpanyl chains from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1

The structures of two novel polar lipids (AGI and AI) of an aerobic hyperthermophilic archaeon, Aeropyrum pernix, were elucidated. AGI and AI were the only two major lipids and accounted for 91 mol% and 9 mol%, respectively, of total polar lipids of this organism. The core lipid of A. pernix total lipids consisted solely of 2,3-di- O-sesterterpanyl- sn-glycerol (C 25,25-archaeol). The molecular weights of the free acid forms of AGI and AI were shown by FAB-mass spectrometry to be 1196 and 1034, respectively. AI was completely hydrolyzed by phosphatidylinositol-specific phospholipase C, while AGI was not hydrolyzed at all under the same condition for the hydrolysis of AI. The molar ratio of phosphate, myo-inositol, and glucose in AGI was 1.0:0.97:0.95. The positions of linkages between myo-inositol and glucose, and between myo-inositol and phosphate in AGI were determined by NMR analyses of intact AGI and glucosylinositol prepared from AGI. Finally, it was concluded that the structures of AGI and AI were 2,3-di- O-sesterterpanyl- sn-glycerol-1-phospho-1′-(2′- O-α- d-glucosyl)- myo-inositol (C 25,25-archaetidyl(glucosyl)inositol) and 2,3-di- O-sesterterpanyl- sn-glycerol-1-phospho- myo-inositol (C 25,25-archaetidylinositol), respectively. This is the first example that a core lipid of whole polar lipids is composed of only one species C 25,25-archaeol in one organism and that glucosylinositol is found in a polar lipid as a polar head group.

Molecular characterization and postsplicing fate of three introns within the single rRNA operon of the hyperthermophilic archaeon Aeropyrum pernix K1.

The single rRNA operon (arnS-arnL) of the hyperthermophilic archaeon Aeropyrum pernix K1 was sequenced. The DNA sequence data and detailed RNA analyses disclosed an unusual feature: the presence of three introns at hitherto undescribed insertion positions within the rRNA genes. The 699-nucleotide (nt) intron Ialpha was located at position 908 (Escherichia coli numbering [H. F. Noller, Annu. Rev. Biochem. 53:119-162, 1984]) of the 16S rRNA, while the 202-nt intron Ibeta and 575-nt intron Igamma were located at positions 1085 and 1927 (E. coli numbering), respectively, of the 23S rRNA. They were located within highly conserved sites which have been implicated as crucial for rRNA function in E. coli. All three introns were remarkably AT rich (41.5 to 43.1 mol% G+C) compared with the mature rRNAs (67.7 and 69.2 mol% G+C for 16S and 23S rRNAs, respectively). No obvious primary sequence similarities were detected among them. After splicing from rRNA transcripts in vivo, a large quantity of intronic RNAs were stably retained in the linear monomeric form, whereas a trace of topoisomeric RNA molecules also appeared, as characterized by their behavior in two-dimensional gel electrophoresis. Secondary structural models of the Ialpha-, Ibeta-, and Igamma-containing rRNA precursors agree with the bulge-helix-bulge motif. Two of the introns, Ialpha and Igamma, contained open reading frames whose protein translation exhibited no overall similarity with proteins reported so far. However, both share a LAGLI-DADG motif characteristic of homing endonucleases.

An extremely heat-stable extracellular proteinase (aeropyrolysin) from the hyperthermophilic archaeon Aeropyrum pernix K1

An extracellular metalloproteinase, which we had designated aeropyrolysin, from the aerobic marine hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820), was purified by ammonium sulfate precipitation, anionic exchange chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 52 kDa as determined by SDS-PAGE. The proteinase had a broad pH optimum (pH 5–9) with a maximal activity at pH 6–8 for azocasein hydrolysis. The optimum temperature for enzyme activity was 100°C in the absence of 1 mM CaCl 2 and 110°C in the presence of 1 mM CaCl 2. The enzyme activity was completely inhibited by EDTA and EGTA, indicating that it was a metalloproteinase. The enzyme was highly resistant to the denaturing reagents urea, guanidine-HCl, dithiothreitol, 2-mercaptoethanol and SDS. The enzyme also showed a high activity with the metalloproteinase specific substrate MOCAc-Pro-Leu-Gly-Leu-A 2pr(Dnp)-Ala-Arg-NH 2. The enzyme was extremely thermostable showing half-lives of 2.5 h at 120°C and 1.2 h at 125°C in the presence of 1 mM CaCl 2. These results indicate that this enzyme is one of the most thermostable extracellular proteinases reported to date.