Functional compartmentalization and metabolic separation in a prokaryotic cell

The prokaryotic cell is traditionally seen as a “bag of enzymes,” yet its organization is much more complex than in this simplified view. By now, various microcompartments encapsulating metabolic enzymes or pathways are known for Bacteria. These microcompartments are usually small, encapsulating and concentrating only a few enzymes, thus protecting the cell from toxic intermediates or preventing unwanted side reactions. The hyperthermophilic, strictly anaerobic Crenarchaeon Ignicoccus hospitalis is an extraordinary organism possessing two membranes, an inner and an energized outer membrane. The outer membrane (termed here outer cytoplasmic membrane) harbors enzymes involved in proton gradient generation and ATP synthesis. These two membranes are separated by an intermembrane compartment, whose function is unknown. Major information processes like DNA replication, RNA synthesis, and protein biosynthesis are located inside the “cytoplasm” or central cytoplasmic compartment. Here, we show by immunogold labeling of ultrathin sections that enzymes involved in autotrophic CO₂ assimilation are located in the intermembrane compartment that we name (now) a peripheric cytoplasmic compartment. This separation may protect DNA and RNA from reactive aldehydes arising in the I. hospitalis carbon metabolism. This compartmentalization of metabolic pathways and information processes is unprecedented in the prokaryotic world, representing a unique example of spatiofunctional compartmentalization in the second domain of life.

Compartmentalization is one of the distinguishing features of eukaryotic cells, which contain membrane-bound organelles in order to perform their specific functions more efficiently, like photosynthesis and CO₂ fixation in plants. Here, autotrophic CO₂ assimilation proceeds via the Calvin–Benson cycle in the stroma of chloroplasts. Evolutionary ancestors of chloroplasts, the cyanobacteria, contain the key carboxylating enzyme of this cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), in specifically insulated proteinaceous microcompartments, carboxysomes, to enrich both the carboxylase and its substrate CO₂ and to exclude competing O₂ at the active site of RubisCO (1). Other enzymes involved in autotrophy are located in the cytoplasm. Besides the Calvin–Benson cycle, autotrophic prokaryotes have developed at least six alternative ways of CO₂ fixation (2–4). The recently discovered autotrophic cycle, the dicarboxylate/4-hydroxybutyrate (DC/HB) cycle (Fig. 1A), functions in the anaerobic hyperthermophilic Crenarchaeota of the orders Desulfurococcales and Thermoproteales, for example, Ignicoccus hospitalis, Thermoproteus neutrophilus, and Pyrolobus fumarii (2, 5–9). In the carbon fixation phase of this pathway, one molecule acetyl-CoA, one molecule CO₂, and one molecule bicarbonate are converted to oxaloacetate via pyruvate synthase and phosphoenolpyruvate (PEP) carboxylase reactions. In the reduction phase, oxaloacetate is reduced to succinyl-CoA and then to 4-hydroxybutyrate, a name-giving intermediate of the cycle. Finally, in the acetyl-CoA regeneration phase of the cycle, 4-hydroxybutyrate is activated to the corresponding CoA-ester, dehydrated to crotonyl-CoA, and converted to two molecules of acetyl-CoA via β-oxidation reactions.Open in a separate windowFig. 1.The DC/HB cycle (adapted from ref. 3) (A), ultrastructure (B), and 3D model (C) of I. hospitalis. Enzymes: 1) pyruvate synthase, 2) pyruvate:water dikinase, 3) PEP carboxylase, 4) malate dehydrogenase, 5) fumarate hydratase, 6) fumarate reductase (natural electron donor is not known), 7) succinyl-CoA synthetase, 8) succinyl-CoA reductase (natural electron acceptor is not known), 9) succinic semialdehyde reductase, 10) 4-hydroxybutyrate-CoA ligase, 11) 4-hydroxybutyryl-CoA dehydratase, 12) crotonyl-CoA hydratase, 13) (S)-3-hydroxybutyryl-CoA dehydrogenase, and 14) acetoacetyl-CoA β-ketothiolase. Fd, ferredoxin; MV, methyl viologen. The enzymes studied in this work are highlighted in red. Ultrathin section (B) and 3D model of a semithin section (C) of a cryo-fixed, freeze-substituted, Epon-embedded cell. CCC (central cytoplasmic compartment), PCC (peripheric cytoplasmic compartment), ICM (inner cytoplasmic membrane), TN (tubular network), OCM (outer cytoplasmic membrane), and Neq (Nanoarchaeum equitans). 3D model highlights the TN originating from the CCC. (Scale bars, 500 nm.)The DC/HB cycle has first been discovered in the hyperthermophilic autotrophic sulfur-reducing archaeon Ignicoccus hospitalis (6) that grows under anaerobic conditions with molecular hydrogen and sulfur at T = 73 to 98 °C (T_opt 90 °C) (10). Apart from the unusual carbon assimilation pathway, Ignicoccus cells exhibit an extraordinary two-membrane ultrastructure (Fig. 1 B and C). The outer cellular membrane (not to be confused with the outer membrane of gram-negative bacteria) encases the intermembrane compartment, while the inner membrane surrounds a modified cytoplasm. Here, we suggest naming these compartments peripheric and central cytoplasmic compartment (CC), respectively, that are enclosed by outer and inner cytoplasmic membranes (CM). The peripheric CC contains a complex tubular network, derived from the central CC (10–13). The spatial compartmentalization goes along with a functional compartmentalization, as energy-conserving ATP synthase and H₂:sulfur oxidoreductase are located in the outer CM, while the major energy-consuming anabolic steps, DNA replication, RNA synthesis, and translation, take place in the central CC (14). Here, we present the subcellular localization of four enzymes of the DC/HB cycle and the biochemical characterization of three of them. Our study shows that autotrophic CO₂ assimilation in I. hospitalis proceeds in the peripheric CC, thus demonstrating another unique feature of I. hospitalis: the spatial separation of major anabolic processes in I. hospitalis, namely DNA replication, RNA synthesis, and protein biosynthesis from inorganic carbon fixation.