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Abstract

The membrane-spanning ferredoxin:NAD+ oxidoreductase (Rnf) complex of the acetogenic bacterium Clostridium ljungdahlii plays a vital role in chemiosmotic ion-gradient formation through an ion-pumping function. The ATP synthase uses the higher outer cell ion-gradient for ATP generation. In previous studies, an increased ATP gain was described under nitrate supplementation. Here, our main interest is the elucidation of an interplay between the Rnf complex and proteins involved in energy conversion. For this purpose, we established an in-vivo interactomic approach for C. ljungdahlii based on affinity purification of protein complexes coupled with mass spectrometry. A strep-tagged version of RnfC from a recombinant clostridial strain was used as a bait protein to capture relevant interaction partners under heterotrophic conditions. The wild-type strain with an empty vector underwent similar experimental conditions and was used as a column control. Furthermore, we performed a proteomic experiment to visualize all of the produced wild- type proteins before purification. Proteins involved in nitrogen and alcohol metabolism as well as in formate production interacted with the Rnf complex. Moreover, the similarly used interactomic approach in the presence of nitrate showed a rearrangement of interaction partners. Next, obtained Rnf complex interactions will be visualized by using polyclonal antibodies against the bait- and prey proteins in an immunogold labeling experiment via transmission electron microscope. Finally, our results showed a high-ordered Rnf complex formation in C. ljungdahlii, which provides an energetic benefit for the cell. A rearrangement of the Rnf supercomplex demonstrated the constant change of a dynamic bacterial cell system to the environment. Overall, the elucidation of energy generation processes in C. ljungdahlii forms the basis for genetic cell modification of this strain to produce an abundance of industry-relevant products.