A study on the CO₂ fixation pathway of Ignicoccus hospitalis

This thesis mainly focuses on biochemical investigation and subcellular localization of enzymes involved in the CO2 fixation pathway (Dicarboxylate/4-Hydroxybutyrate pathway) of the hyperthermophilic Archaeon Ignicoccus hospitalis. A further objective was to compare different methods in electron microscopy to optimize immunogold labeling on ultrathin sections of I. hospitalis. Comparing immunogold labeling on ultrathin sectioned I. hospitalis cells, embedded in different resins (Epon versus Lowicryl) and on cryo-sections, demonstrated that the three different methods lead to essentially similar labeling patterns. Due to excellent preservation of ultrastructure, immuno electron microscopy on Epon sections proved to be the method of choice for Ignicoccus cells. Utilizing an accelerated protocol for immunogold labeling clearly demonstrated that shorter incubation times for antibodies are fully sufficient and contribute to better maintenance of ultrastructural details. To confirm the so far only putative I. hospitalis genes of the carbon fixation cycle, candidates for enzymes involved in that pathway were investigated biochemically: Igni_0341 (PEP carboxylase), Igni_1263 (malate dehydrogenase), Igni_0379 and Igni_0475 (4-hydroxybutyryl-CoA synthetase) and Igni_1058 (crotonyl-CoA hydratase/3-hydroxybutyryl-CoA dehydrogenase). Additionally, a candidate gene for a succinic semialdehyde reductase, a 'missing' enzyme of the cycle, was detected in the genome of I. hospitalis and heterologously overexpressed in E. coli. Based on enzyme assays and bioinformatics, the recombinant 'candidate' enzymes Igni_1263, Igni_0132 and Igni_1058 could clearly be identified as the respective enzymes involved in the carbon fixation pathway of I. hospitalis. Immuno electron microscopy revealed a localization of PEP carboxylase, malate dehydrogenase, succinic semialdehyde reductase and crotonyl- CoA hydratase/3-hydroxybutyryl-CoA dehydrogenase in the intermembrane compartment of I. hospitalis, tightly associated with the outer cellular membrane. From these results it was assumed that the whole CO2 fixation pathway of I. hospitalis takes place in the intermembrane compartment of the organism, not in the cytoplasm. Furthermore, genes encoding for enzymes of the dicarboxylate/4-hydroxybutyrate cycle were detected in the genome of I. islandicus. Malate dehydrogenase and succinic semialdehyde reductase of I. islandicus were also shown to be located in the intermembrane compartment. Similar as demonstrated for I. hospitalis, these results strongly indicate that CO2 fixation of I. islandicus takes place in the intermembrane compartment according to the dicarboxylate/4-hydroxybutyrate pathway