Cytotoxicity and uptake of archaeosomes prepared from Aeropyrum pernix lipids.

Archaeon Aeropyrum pernix K1 is an obligate aerobic hyperthermophilic organism with C25,25-archeol membrane lipids with head groups containing inositol. Interactions of archaeosomes, liposomes prepared from lipids of A. pernix, with mammalian cells in vitro were studied. In vitro cytotoxicity was tested on five different cell lines: rodent mouse melanoma cells (B16-F1) and Chinese hamster ovary (CHO) cells, and three human cell lines-epithelial colorectal adenocarcinoma cells (CACO-2), liver hepatocellular carcinoma cell line (Hep G2) and endothelial umbilical vein cell line (EA.hy926). Archaeosomes were nontoxic to human Hep G2, CACO-2 and mildly toxic to rodent CHO and B16-F1 cells but showed strong cytotoxic effect on EA.hy926 cells. Confocal microscopy revealed that archaeosomes are taken up by endocytosis. The uptake of archaeosomes and the release of loaded calcein are more prominent in EA.hy926 cells, which is in line with high toxicity toward these cells. The mechanisms of uptake, release and action in these cells as well as in vivo functioning have to be further studied for possible targeted drug delivery.

Effect of growth medium pH of Aeropyrum pernix on structural properties and fluidity of archaeosomes.

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.

In vivo characterization of thermal stabilities of Aeropyrum pernix cellular components by differential scanning calorimetry.

Revival studies of Aeropyrum pernix show that the viability of cells and cell recovery after heat treatment depends on the temperature of treatment. Differential scanning calorimetry (DSC) is used to analyze the relative thermal stabilities of cellular components of A. pernix and to identify the cellular components responsible for the observed lag phase and reduced maximum growth following a heat treatment. DSC thermograms show 5 visible endothermic transitions with 2 major transitions. DSC analysis of isolated crude ribosomes aids the assignment of the 2 major peaks observed in whole-cell thermograms to denaturation of ribosomal structures. A comparison of partial and immediate full rescan thermograms of A. pernix whole cells indicates that both major peaks represent irreversible thermal transitions. A DNA peak is also identified in the whole-cell thermogram by comparison with the optical data of isolated pure DNA. DNA melting is shown to be irreversible in dilute solution, whereas it is partially reversible in whole cells, owing at least in part, to restricted volume effects. In contrast to mesophilic organisms, hyperthermophilic A. pernix ribosomes are more thermally stable than DNA, but in both organisms, irreversible changes leading to cell death occur owing to ribosomal denaturation.

Characterization of thermostable native alkaline phosphatase from an aerobic hyperthermophilic archaeon, Aeropyrum pernix K1.

This paper reports the characterization of an alkaline phosphatase (AP) from an aerobic hyperthermophilic Archaeon Aeropyrum pernix K1. The native AP was purified into homogeneity. The enzyme is predicted as a homodimeric structure with a native molecular mass of about 75 kDa and monomer of about 40 kDa. Apparent optimum pH and temperature were estimated at 10.0 and above 95 degrees C, respectively. Magnesium ion increased both the stability and the activity of the enzyme. A. pernix AP has been demonstrated as a very thermostable AP, retaining about 76% of its activity after being incubated at 90 degrees C for 5.5 h and 67% of its activity after being incubated at 100 degrees C for 2.5 h, respectively, under the presence of Mg(II). Enzyme activity was increased in addition of exogenous Mg(II), Ca(II), Zn(II), and Co(II).

Identification of the first archaeal oligopeptide-binding protein from the hyperthermophile Aeropyrum pernix.

The archaeon Aeropyrum pernix grows optimally at 90 degrees C and derives energy primarily from aerobic degradation of complex proteinaceous substrates. The ability of these nutrients to sustain growth is generally associated with the presence of oligopeptide transport systems, such as the well-known protein-dependent ATP-binding cassette (ABC) transporters. This study is concerned with the isolation and characterisation of the first archaeal oligopeptide-binding protein (OppA(Ap)) from the extracellular medium of A. pernix. The protein shows a pI of 3.9 and a molecular mass of about 90 kDa under native conditions. By using a proteomic approach, the OppA(Ap)-encoding gene was identified (APE1583) and about 55% of the protein amino-acid sequence was validated. The extracellular purified protein was able to efficiently bind oligopeptide substrates such as Xenopsin. The amount of a liganded peptide to OppA(Ap) was about 70% at 90 degrees C using a 1/100 (w/w) OppA(Ap)/substrate ratio. Sequence comparisons showed a weak but significant similarity of OppA(Ap) with bacterial oligopeptide binding proteins. Furthermore, APE1583 neighbouring genes encode for the cognate components of an ABC transport system, suggesting that these ORFs are organised in an operon-like structure, with OppA(Ap )as the extracellular component for the uptake of oligopeptides.

Optimization of growth for the hyperthermophilic archaeon Aeropyrum pernix on a small-batch scale.

Growth of Aeropyrum pernix, the first reported aerobic neutrophilic hyperthermophilic archaeon, was investigated under different cultivation parameters. Different sources of seawater, pH, and the cultivation methods were tested with the aim to improve the biomass production. A 1-L glass flask fitted with a condenser and air diffuser was used as a bioreactor. The optimum conditions for maximizing A. pernix biomass were obtained when Na2S2O3.5H2O (1 g/L) with added marine broth 2216 at pH 7.0 (20 mmol HEPES buffer/L) was used as a growing medium in a 1-L flask. The biomass production was 0.45 g dry cell mass/L in 40 h under the optimum conditions, which is more than the 0.42 g dry cell mass/L in 60 h previously obtained.

Aeropyrum camini sp. nov., a strictly aerobic, hyperthermophilic archaeon from a deep-sea hydrothermal vent chimney.

A novel hyperthermophilic archaeon, designated strain SY1(T), was isolated from a deep-sea hydrothermal vent chimney sample collected from the Suiyo Seamount in the Izu-Bonin Arc, Japan, at a depth of 1385 m. The cells were irregular cocci (1.2 to 2.1 micro m in diameter), occurring singly or in pairs, and stained Gram-negative. Growth was observed between 70 and 97 degrees C (optimum, 85 degrees C; 220 min doubling time), pH 6.5 and 8.8 (optimum, pH 8.0), and salinity of 2.2 and 5.3 % (optimum, 3.5 %). It was a strictly aerobic heterotroph capable of growing on complex proteinaceous substrates such as yeast extract and tryptone. The G+C content of the genomic DNA was 54.4 mol%. Phylogenetic analysis based on the 16S rDNA sequence of the isolate indicated that the isolate was closely related to Aeropyrum pernix strain K1(T). However, no significant genetic relatedness was observed between them by DNA-DNA hybridization. On the basis of the molecular and physiological traits of the new isolate, the name Aeropyrum camini sp. nov. is proposed, with the type strain SY1(T) (=JCM 12091(T)=ATCC BAA-758(T)).