Energized outer membrane and spatial separation of metabolic processes in the hyperthermophilic Archaeon Ignicoccus hospitalis - PubMed (original) (raw)

Energized outer membrane and spatial separation of metabolic processes in the hyperthermophilic Archaeon Ignicoccus hospitalis

Ulf Küper et al. Proc Natl Acad Sci U S A. 2010.

Abstract

ATP synthase catalyzes ATP synthesis at the expense of an electrochemical ion gradient across a membrane that can be generated by different exergonic reactions. Sulfur reduction is the main energy-yielding reaction in the hyperthermophilic strictly anaerobic Crenarchaeon Ignicoccus hospitalis. This organism is unusual in having an inner and an outer membrane that are separated by a huge intermembrane compartment. Here we show, on the basis of immuno-EM analyses of ultrathin sections and immunofluorescence experiments with whole I. hospitalis cells, that the ATP synthase and H(2):sulfur oxidoreductase complexes of this organism are located in the outer membrane. These two enzyme complexes are mandatory for the generation of an electrochemical gradient and for ATP synthesis. Thus, among all prokaryotes possessing two membranes in their cell envelope (including Planctomycetes, gram-negative bacteria), I. hospitalis is a unique organism, with an energized outer membrane and ATP synthesis within the periplasmic space. In addition, DAPI staining and EM analyses showed that DNA and ribosomes are localized in the cytoplasm, leading to the conclusion that in I. hospitalis energy conservation is separated from information processing and protein biosynthesis. This raises questions regarding the function of the two membranes, the interaction between these compartments, and the general definition of a cytoplasmic membrane.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Detection of ATP synthase by ATP hydrolysis in-gel assay and Western blotting. (A) ATP hydrolysis activity test (90 min at 80 °C) after native protein complex separation (hrCNE) of the I. hospitalis solubilisate. (B) Immunoblot of an hrCNE gel using the 440 kDa complex antibody of I. hospitalis (dilution 1:5,000). (C) Immunoblot of an SDS/PAGE gel using both antibodies raised against the A and B subunits of M. jannaschii (dilution 1:10,000). Immunolabeling was visualized by HRP-conjugated secondary antibodies (Sigma-Aldrich; dilution, 1:5,000).

Fig. 2.

Localization of A1AO ATP synthase on I. hospitalis cells by EM of ultrathin sections. (A) Labeling with antibodies specifically raised against the purified 440-kDa ATPase complex, (B) Labeling with antibodies raised against the membrane-bound subunit a. For both images the secondary antibody with ultrasmall gold particles is visualized by silver enhancement (dilution, 1:200). Dilution of the primary antibodies, 1:200. C, cytoplasm; IM, inner membrane; V, vesicles in the periplasm; OM, outer membrane. (Scale bars, 1 μm.)

Fig. 3.

Epifluorescence microphotographs of I. hospitalis cells. (A) Phase contrast image. (B) Merged image of phase contrast and DAPI-stained cytoplasm (blue). (C) Localization of ATP synthase immunolabeled with the specific 440-kDa ATPase complex antibody; secondary antibody coupled to Alexa Fluor 488 (green). (D) Merge of Fig. 2 B and C. (E) Merge of a phase contrast image and an immunofluorescence image of a dividing I. hospitalis cell. (F) Scheme of the dividing cell in Fig. 2_E_. (Scale bars, 2 μm.)

Fig. 4.

Localization of H2:sulfur oxidoreductase complex on I. hospitalis cells with antibodies specifically raised against the purified complex of P. abyssi strain TAG11. (A) EM of ultrathin sections: the immunolabeling was performed similar to Fig. 2; C, cytoplasm; IM, inner membrane; V, vesicles in the periplasm; OM, outer membrane. (Scale bar, 1 μm.) (B) Merged image of phase contrast and DAPI-stained cytoplasm (blue). (C) Colocalization of H2:sulfur oxidoreductase complex and cytoplasm visualized by immunofluorescence (secondary antibody coupled to Alexa Fluor 488, green) and DAPI staining. (Scale bars in B and C, 2 μm.)

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Energized outer membrane and spatial separation of metabolic processes in the hyperthermophilic Archaeon Ignicoccus hospitalis - PubMed (original) (raw)