Energy conservation in the Ech-containing thermophilic acetogenic bacterium Thermoanaerobacter kivui

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Acetogenic microorganisms are defined by their ability to grow with H2 as electron donor, reducing CO2 to acetic acid via the Wood-Ljungdahl pathway, the presumably oldest CO2 fixation pathway. It has been an enigma for decades how acetogens conserve energy, but was recently unravelled for the model acetogen A. woodii. This acetogen has been found to have a respiratory chain consisting of a membrane bound Na+ translocating ferredoxin:NAD oxidoreductase, the Rnf complex, and a sodium ion-dependent ATP synthase. However, many acetogenic bacteria are devoid of rnf genes. A genomic perspective revealed that rnf-free acetogens have genes encoding energy-converting hydrogenases (ech) instead (some may have both). The latter encode multi-subunit, membrane-integral, electron transfer protein complexes. Indirect evidence, mostly gathered for archaea, together with their evolutionary relatedness to complex I of the respiratory chain are taken as indication that Ech complexes are respiratory enzymes that couple electron transfer from a donor such as reduced ferredoxin to protons (under liberation of hydrogen) with the translocation of ions across the cytoplasmic membrane. Thus, it is assumed that they create an electrochemical ion gradient that is then used by an ATP synthase to synthesize ATP. The hypothesis that the respiratory chain in rnf-free, ech-containing acetogens consists of a proton/sodium ion-translocating, proton-reducing Ech hydrogenase will be addressed in the thermophilic acetogenic bacterium Thermoanaerobacter kivui. We will use a two-pronged approach to obtain a complete picture of the energy metabolism of T. kivui using a combination of physiological/biochemical studies and genetic analyses. Interestingly, the T. kivui genome encodes for two Ech complexes. We will develop a protocol for the purification of Ech complex(es) from T. kivui using different chromatographic steps under strictly anaerobic conditions. The subunit composition and biochemical properties of the enzyme complex will be determined. Of special interest is a potential sodium ion dependence of the electron transfer. The final goal is to incorporate the enzyme in liposomes, to proof that Ech is an electron transfer-driven H+ / Na+ pump and to characterize ion translocation by Ech in these proteoliposomes. Moreover, we will apply methods for genetic manipulations on the T. kivui genome recently established in our lab to generate chromosomal deletions of genes encoding Ech1, Ech2, subunits of both Ech complexes or both Ech complexes simultaneously. The physiology of the deletion mutants will be studied under different growth conditions. Subsequently, energy conservation will be studied in inverted membrane vesicles. This will answer the question about the role of Ech complexes in physiology and energy conservation in T. kivui.