The mechanisms by which DNA is taken up into the bacterial cell during natural genetic transformation are poorly understood. Although related components essential to the uptake of DNA during transformation have been defined in Gram-negative species, it remains unclear whether DNA binding and uptake are dissociable events. Therefore, DNA uptake has been the earliest definable step in any Gram-negative transformation pathway. In the human pathogen Neisseria gonorrhoeae, sequence-specific DNA uptake requires an intact type IV pili (Tfp) biogenesis machinery along with three molecules that are dispensable for Tfp expression: ComP (a pilin subunit-like molecule), PilT (a cytoplasmic protein involved in pilus retraction) and ComE (a periplasmic protein with intrinsic DNA-binding activity). By conditionally altering the levels of ComP and PilT expression, we show here that DNA binding and uptake are resolvable events. Consequently, we are able to demonstrate that PilT is largely dispensable for functional DNA binding and, therefore, contributes specifically to uptake. Furthermore, sequence specificity in this system is imposed at the level of DNA binding, a process that is influenced by both ComP and PilE. However, sequence-specific DNA binding is not attributable to an intrinsic property of the Tfp subunit protein. Finally, we demonstrate the existence of a robust, non-specific DNA-binding activity associated with the expression of both Tfp and PilT, which is unrelated to transformation but obscures the observation of specific binding events.