ATP synthase is a complex universal enzyme responsible for ATP synthesis across all kingdoms of life. The F-type ATP
synthase has been suggested to have evolved from two functionally independent, catalytic (F1) and membrane bound (Fo),
ancestral modules. While the modular evolution of the synthase is supported by studies indicating independent assembly of
the two subunits, the presence of intermediate assembly products suggests a more complex evolutionary process. We analyzed
the phylogenetic profiles of the human mitochondrial proteins and bacterial transcription units to gain additional insight into
the evolution of the F-type ATP synthase complex. In this study, we report the presence of intermediary modules based on the
phylogenetic profiles of the human mitochondrial proteins. The two main intermediary modules comprise the α3β3 hexamer
in the F1 and the c-subunit ring in the Fo. A comprehensive analysis of bacterial transcription units of F1Fo ATP synthase
revealed that while a long and constant order of F1Fo ATP synthase genes exists in a majority of bacterial genomes, highly
conserved combinations of separate transcription units are present among certain bacterial classes and phyla. Based on our
findings, we propose a model that includes the involvement of multiple modules in the evolution of F1Fo ATP synthase. The
central and peripheral stalk subunits provide a link for the integration of the F1/Fo modules.