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Energized Outer Membrane and Spatial Separation of Metabolic Processes in the Hyperthermophilic Archaeon Ignicoccus Hospitalis

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Energized Outer Membrane and Spatial Separation of Metabolic Processes in the Hyperthermophilic Archaeon Ignicoccus Hospitalis Energized outer membrane and spatial separation of metabolic processes in the hyperthermophilic Archaeon Ignicoccus hospitalis Ulf Küpera, Carolin Meyerb, Volker Müllerc, Reinhard Rachelb, and Harald Hubera,1 aInstitute for Microbiology and Archaeal Center, Universität Regensburg, D-93053 Regensburg, Germany; bCenter for Electron Microscopy, Faculty of Natural Sciences III, Universität Regensburg, D-93053 Regensburg, Germany; and cMolecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe Universität Frankfurt, D-60438 Frankfurt am Main, Germany Edited* by Dieter Söll, Yale University, New Haven, CT, and approved December 18, 2009 (received for review October 9, 2009) ATP synthase catalyzes ATP synthesis at the expense of an electro- To date, in prokaryotes no outer membranes but only cytoplasmic chemical ion gradient across a membrane that can be generated by membranes have been described as harboring ATP synthase com- different exergonic reactions. Sulfur reduction is the main energy- plexes, the key components in cellular bioenergetics (15). These yielding reaction in the hyperthermophilic strictly anaerobic Cren- complexes (bacteria, mitochondria, and chloroplasts: F1FO ATP archaeon Ignicoccus hospitalis. This organism is unusual in having an synthases; Archaea: A1AO ATP synthases) consist of a hydrophilic inner and an outer membrane that are separated by a huge inter- (F1,A1) and a membrane-bound domain (FO,AO) (16). Driven by membrane compartment. Here we show, on the basis of immuno-EM an electrochemical ion gradient (17), the membrane-bound domain analyses of ultrathin sections and immunofluorescence experiments translocates ions (H+;Na+) across the membrane, resulting in ATP with whole I. hospitalis cells, that the ATP synthase and H2:sulfur synthesis by the hydrophilic, catalytic domain. The enzyme is also oxidoreductase complexes of this organism are located in the outer able to reverse this process by hydrolyzing ATP. In contrast to other membrane. These two enzyme complexes are mandatory for the ATP hydrolyzing enzymes, this complex is sensitive to specific generation of an electrochemical gradient and for ATP synthesis. inhibitors. According to the genome annotation of I. hospitalis, Thus, among all prokaryotes possessing two membranes in their cell several ATP hydrolyzing enzymes are present (18); only one set of envelope (including Planctomycetes, Gram-negative bacteria), I. hos- subunits, however, was predicted (A, B, C, D, E, F, a,andc)tobuild MICROBIOLOGY pitalis is a unique organism, with an energized outer membrane and a functional ATP synthase (19) and thought to be located in the ATP synthesis within the periplasmic space. In addition, DAPI stain- inner membrane of I. hospitalis (18). The latter assumption was also ing and EM analyses showed that DNA and ribosomes are localized in based on the fact that primary H+ or Na+ pumps are absent in outer the cytoplasm, leading to the conclusion that in I. hospitalis energy membranes of mitochondria, chloroplasts, and Gram-negative conservation is separated from information processing and protein bacteria (12, 20), so that a gradient sufficient to drive ATP synthesis biosynthesis. This raises questions regarding the function of the two cannot be generated. Therefore, outer membranes are generally membranes, the interaction between these compartments, and the believed to be “non–energy-conserving” (13). To date, neither a general definition of a cytoplasmic membrane. proton motive force across an outer membrane nor ATP synthesis within a periplasmic space has been described. Archaea | ATP synthase | ATPase | immunolabeling | sulfur reductase In this article we show that in I. hospitalis the outer membrane is energized and that ATP synthesis is spatially separated from DNA he hyperthermophilic Crenarchaeon Ignicoccus hospitalis replication, transcription, and protein biosynthesis. These results T TKIN4/I is a strictly anaerobic chemolithoautotrophic sulfur raise questions regarding the function of the two membranes in reducer that grows optimally at 90 °C. It conserves energy by the I. hospitalis, the interaction between the two cell compartments, the reduction of elemental sulfur with molecular hydrogen and uses general definition of a cytoplasmic membrane, and a possible energy CO2 as sole carbon source (1). Together with Nanoarchaeum transfer from I. hospitalis to N. equitans. equitans,itformsan“intimate association,” the only known stable coculture of two Archaea (2, 3). Like all Ignicoccus species (4), I. Results hospitalis cells possess an unusual architecture, with two compart- Purification and Identification of the 440-kDa Subcomplex of ATP ments that can clearly be distinguished in composition and mor- Synthase. To clarify how I. hospitalis conserves energy, we started to phologic appearance. As shown in a number of EM studies (5–7), purify and characterize its A1AO ATP synthase. We solubilized the densely packed cytoplasm is surrounded by two membranes, an membrane proteins of I. hospitalis by addition of n-dodecyl-β-D- “ ” “ ” inner membrane and an outer membrane. These two mem- maltopyranoside (DDM). The solubilisate exhibited a specificATP branes enclose an intermembrane compartment with a variable hydrolysis activity of 1.7 U/mg protein. This activity was completely width from 20 to 500 nm, resulting in a volume exceeding that of the inhibited by the addition of diethylstilbestrol (DES, 1.5 mM) and to cytoplasm (5). Its low electron density suggests that it is devoid of approximately 40% by N′,N′-dicyclohexylcarboiimide (DCCD, 1.5 cellular material like ribosomes or DNA, and it was therefore mM), a property characteristic for a coupled A A ATP synthase “ ” 1 O named periplasm (7). The inner membrane, called the cytoplas- complex. After separating the solubilisate by high-resolution mic membrane, releases numerous vesicles into the periplasmic clear native electrophoresis (hrCNE), the protein complexes were space and also engulfs vesicles into the cytoplasm (7). Both mem- branes exhibit similar lipid composition, with the exception that the outer membrane lacks caldarchaeol cores (8). In addition, the latter Author contributions: U.K., V.M., R.R., and H.H. designed research; U.K. and C.M. per- contains multiple copies of a pore-forming complex (9), whereas a formed research; U.K., C.M., V.M., R.R., and H.H. analyzed data; and U.K., V.M., R.R., and surface layer (S-layer), typical for most Crenarchaeota, is lacking H.H. wrote the paper. (10). Therefore, the architecture of the I. hospitalis cell envelope is The authors declare no conflict of interest. unique among Archaea. Moreover, owing to its huge intermem- *This Direct Submission article had a prearranged editor. brane compartment and an outer membrane without LPS and 1To whom correspondence should be addressed. E-mail: [email protected]. – porins (11 14), it is fundamentally different from other prokaryotic This article contains supporting information online at www.pnas.org/cgi/content/full/ cell envelopes with two membranes (e.g., Gram-negative bacteria). 0911711107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0911711107 PNAS Early Edition | 1of5 Downloaded by guest on September 29, 2021 checked for their ability to hydrolyze ATP in an in-gel enzymatic assay (Fig. 1A). One prominent active complex with an apparent molecular mass of ≈440 kDa was obtained. This complex was purified by ion exchange chromatography, ultrafiltration, and size exclusion chromatography. After tryptic digestion, MALDI-TOF analysis revealed two subunits, A and B, to be part of the I. hospitalis ATPase (scores 577 and 316, respectively). Using specific antibodies generated against the complex, Western blot analyses after hrCNE gave a single signal at an apparent mass of 440 kDa for the native complex (Fig.1B).After its extraction and separation ofthe subunits under denaturing conditions (SDS/PAGE), Western blot analysis showed that the masses of the major subunits A and B (approx- imately 65 and 55 kDa; Fig. 1C), correspond to the predicted masses of the annotated A and B subunits of the ATPase (67 and 52 kDa). Localization of ATP synthase in I. hospitalis. By EM of ultrathin sec- tions of I. hospitalis cells, we investigated the subcellular localization of ATP synthase. Surprisingly, immunolabeling with the antibody raised against the purified 440-kDa ATPase complex showed a highly predominant labeling of the outer membrane of I. hospitalis cells (Fig. 2A). The same result was obtained with antibodies raised against the heterologous membrane-bound subunit a (Fig. 2B)and against the catalytic subunit A of Methanocaldococcus jannaschii (Fig. S1). In all cases, less than 10% of the signals could be detected within the cytoplasm, the inner membrane, and the periplasmic space (including the membrane-coated vesicles). This clearly indicates that thebyfarmajorpartofATPsynthasemoleculesofI. hospitalis is located in the outer membrane. To confirm this result, we carried out fluorescence light microscopy experiments (Fig. 3 A–E)usingDNA staining with DAPI (Fig. 3B) and concomitantly the antibody against the 440-kDa ATPase complex (Fig. 3C). According to these images the DNA is, as expected, exclusively located inside the inner mem- Fig. 1. Detection of ATP synthase by ATP hydrolysis in-gel assay and brane (i.e., in the cytoplasm), whereas we observed the fluorescence Western blotting. (A) ATP hydrolysis activity test (90 min at 80 °C) after of the antibodies bound to the A1AO ATP
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