AMP-forming acetyl coenzyme A synthetase in the outermost membrane of the hyperthermophilic crenarchaeon Ignicoccus hospitalis

Abstract

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic crenarchaeon was found to possess a new CO(2) fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl coenzyme A (acetyl-CoA), which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by two-dimensional native PAGE/SDS-PAGE, followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry and N-terminal sequencing. The ACS of I. hospitalis exhibits a molecular mass of ∼690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immunolabeling on cells of I. hospitalis and other described Ignicoccus species revealed that the ACS is localized at the outermost membrane. This perfectly coincides with recent results that the ATP synthase and the H(2):sulfur oxidoreductase complexes are also located in the outermost membrane of I. hospitalis. These results imply that the intermembrane compartment of I. hospitalis is not only the site of ATP synthesis but may also be involved in the primary steps of CO(2) fixation.

Fig 1

Native membrane protein and subunit composition. (A) In the nondenaturing electrophoresis, hrCNE, of fraction F4 (solubilized membrane and membrane-associated protein complexes) the two high-molecular-mass complexes are obvious which lead to the further analyzed complexes 1 (thermosome) and 2 (ACS). (B) SDS-PAGE of the complexes 1 and 2 from Fig. 1A. Molecular masses are indicated at the left in kilodaltons.

Fig 2

Nondenaturing electrophoresis, hrCNE, of fraction F4 and Western blot. The antibody generated against complex 2 of Fig. 1A was used in this blot. Only one positive reaction at an apparent molecular mass of ∼670 kDa was obtained. St, molecular masses in kilodaltons, are indicated at the left. Lane 1, hrCNE stained with Coomassie blue; lane 2, Western blot.

Fig 3

Acetate consumption during growth of I. hospitalis. The figure shows the decrease of the acetate concentration in the medium during mixotrophic growth with 0.05% yeast extract (■) in relation to the cell concentration (▲). On the other hand, no significant acetate production was detectable under autotrophic culture conditions (▼).

Fig 4

Acetyl-CoA formation by different cell fractions of I. hospitalis. Nearly identical amounts of acetyl-CoA per mg of protein were formed in the crude extract (■) and in isolated membranes (▼), while no significant formation was observed in the cytoplasmic fraction (▲).

Fig 5

Localization of the ACS in the outermost membrane of I. hospitalis by immunoelectron microscopy (two representative examples [A and B]). Sections of high-pressure frozen, freeze-substituted, and Epon-embedded cells were incubated with anti-ACS (primary antibody; dilution, 1:100) and a 6-nm gold-labeled secondary antibody (dilution 1:50) and then visualized by transmission electron microscopy. C, cytoplasm; IM, inner membrane; OM, outermost membrane; IMC, intermembrane compartment. Bars, 200 nm.

Fig 6

Epifluorescence microphotographs of cells from different Ignicoccus species. Bars, 1.5 μm. (A, E, I, and M) Phase-contrast images. (B, F, J, and N) Merged phase-contrast images and SYTO9 fluorescence signals (DNA stain; green). (C, G, K, and O) The localization of the ACS was elucidated by homologous primary antibodies, which were detected by a secondary antibody coupled to Alexa Fluor 546 (red). (D, H, L, and P) Merge of SYTO9 and Alexa Fluor 546 signals.

Fig 7

Scheme of ATP conservation and ATP consumption in the IMC of I. hospitalis.