INACTIVATION OF FHUA AT THE CELL-SURFACE OF ESCHERICHIA-COLI K-12 BY A PHAGE T5 LIPOPROTEIN AT THE PERIPLASMIC FACE OF THE OUTER-MEMBRANE

Inactivation of phage T5 by lysed cells after phage multiplication is prevented by a phage-encoded lipoprotein (Lip) that inactivates the Fh uA outer membrane receptor protein (K. Decker, V. Krauel, A. Meesmann, and K. Heller, Mel. Microbiol. 12:321-332, 1994). Using FhuA derivati ves carrying insertions of 4 and 16 amino acid residues and point muta tions, we determined whether FhuA inactivation is caused by binding of Llp to FhuA and which regions of FhuA are important for inactivation by Llp. Cells expressing Llp were resistant not only to phage T5 but t o all FhuA ligands tested, such as phage phi 80, colicin M, and albomy cin, and they were strongly reduced in the uptake of ferrichrome. Most of the FhuA derivatives which were not affected by Llp were, accordin g to a previously published FhuA transmembrane topology model, located in periplasmic turns and in the TonB box close to the periplasm. Sinc e the ligands bind to the cell surface, interaction of FhuA with Llp i n the periplasm may induce a FhuA conformation which impairs binding o f the ligands. This conclusion was supported by the increase rather th an decrease of colicin M sensitivity of two mutants in the presence of Llp. The only Llp-resistant FhuA derivatives with mutations at the ce ll surface contained insertions of 16 residues in the loop that determ ines the permeability of the FhuA channel and selves as the principal binding site for all FhuA ligands. This region may be inactivated by s teric hindrance in that a portion of Llp penetrates into the channel. Outer membranes prepared with 0.25% Triton X-100 from cells expressing Llp contained inactivated FhuA, suggesting Llp to be an outer membran e protein whose interaction with FhuA nas not abolished by Triton X-10 0. Lip solubilized in 1.1% octylglucoside prevented T5 inactivation by FhuA dissolved in octylglucoside.