This study demonstrates that in Staphylococcus aureus grown under strong oxygen limitation, the regulator ComK2 represses competence development for natural transformation, acting through a direct interaction with the protein SA2107. To confirm this protein–protein interaction, Flow Induced Dispersion Analysis (FIDA) was used to measure the apparent hydrodynamic radius (Rh) of fluorescently labelled SA2107 on its own and mixed with ComK2. On a Fida 1 instrument, FIDA measured SA2107's Rh at 1.97 nm, increasing to 2.75 nm upon complex formation — values closely matching AlphaFold models. A titration gave a dissociation constant (Kd) of 420 nM, establishing that the ComK2–SA2107 interaction is binary and direct. FIDA was one of several complementary techniques supporting the study's genetic and structural findings.