Loop Deletions Indicate Regions Important for FhuA Transport and Receptor Functions in Escherichia coli

Abstract

Precise deletions of cell surface-exposed loops of FhuA resulted in mutants of Escherichia coli with distinct phenotypes. Deletion of loop 3 or 11 inactivated ferrichrome transport activity. Deletion of loop 8 inactivated receptor activity for colicin M and the phages T1, T5, and 80. The loop 7 deletion mutant was colicin M resistant but fully phage sensitive. The loop 4 deletion mutant was resistant to the TonB-dependent phages T1 and 80 but fully sensitive to the TonB-independent phage T5. The phenotypes of the deletion mutants revealed important sites for the multiple FhuA transport and receptor activities. The ligand binding sites are nonidentical and are distributed among the entire exposed surface. Presumably, FhuA evolved as a ferrichrome transporter and was subsequently used as a receptor by the phages and colicin M, which selected the same as well as distinct loops as receptor sites.

The iron–siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11–Pro16 β-hairpin region in the recognition mechanism

“…loops L5, L7, L8 and L11 are essential for MccJ25 activity, and that loop L8 can be considered as the major and maybe the single binding site for phage T5 [16]. Together with this finding, the ability of MccJ25 to prevent the FhuA-phage T5 interaction in our in vivo and in vitro assays suggests that MccJ25 and phage T5 bind to the same loop L8 on the FhuA receptor.…”

“…The binding sites for the different FhuA ligands are located on the external loops of the barrel [16]. The involvement of FhuA in susceptibility to MccJ25 was supported by the observation that a FhuA-deficient E. coli strain resistant to MccJ25 was rendered susceptible upon transfection with a plasmid encoding the E. coli or Salmonella enteritidis Paratyphi FhuA protein, but not when plasmids encoding S. enteritidis Typhimurium or Pantoea agglomerans (previously Erwinia herbicola) FhuA were used [17].…”

“…It exhibits a side-chain-to-backbone cyclization involving Glu 8 and the N-terminal glycine, and adopts a lasso-structure in which the Cterminal end of the peptide is threaded into the cyclic backbone [3]. We have shown recently that the two-chain analogue t-MccJ25 (initially called MccJ25-L [2]), obtained by cleavage with thermolysin, retains the lasso-structure of MccJ25 but loses the Val 11 -Pro 16 hairpin-like structure targeted by the enzyme [4] (Figure 1). Interestingly, the highly potent activity of MccJ25 decreases markedly, but is not fully abrogated, upon thermolysin cleavage [5], indicating that the targeted region is required for complete antibacterial activity.…”

Defined Inactive FecA Derivatives Mutated in Functional Domains of the Outer Membrane Transport and Signaling Protein of Escherichia coli K-12

“…Loop 7 and 8 deletion mutants of FepA display no ferric enterobactin binding and transport (37). FecA and FepA are similar in that deletion of loop 7 or 8 abolishes all activities; in contrast, FhuA loop 7 or 8 deletion mutants retain all activities (15). FecA is similar to FhuA in that deletion of loop 3 or 11 abolishes all activities; in contrast, a loop 3 deletion mutant of FepA retains fully ferric enterobactin transport and sensitivity to colicin B (37).…”

“…The negatively charged dinuclear ferric citrate is probably most strongly bound by the positively charged R365 and R380 residues, suggesting decreased binding of ferric citrate to the R365A and R380A mutants. However, weak binding does not necessarily lead to slow transport, as examples of FhuA (15) and FepA (37) demonstrate. Replacement of R81, which is highly conserved in FhuA homologues, leads to a very strong reduction in binding but fully retained transport (14).…”