The role of the outer-membrane
iron transporter FhuA as a potential receptor for the
antimicrobial peptide MccJ25 (
microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA(-) strain and by using isogenic strains mutated in genes encoding the
protein complex respectively. In addition,
MccJ25 was shown to block phage T5
infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that
MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as
MccJ25 inhibited phage T5
DNA ejection triggered by purified FhuA. Direct interaction of
MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry.
MccJ25 bound to FhuA with a 2:1 stoichiometry and a K(d) of 1.2 microM. Taken together, our results demonstrate that FhuA is the receptor for
MccJ25 and that the
ligand-receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that
MccJ25 binding involves external loops of FhuA. Unlike native
MccJ25, a
thermolysin-cleaved
MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5
infection of E. coli. Therefore the Val11-Pro16 beta-hairpin region of
MccJ25, which is disrupted upon cleavage by
thermolysin, is required for
microcin recognition.