Escherichia coli periplasmic protein FepB binds ferrienterobactin

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• High-affinity iron uptake in Gram-negative bacteria typically involves periplasmic-binding-protein-dependent transport.
• FepB is a periplasmic protein in Escherichia coli essential for iron uptake via the siderophore enterobactin (Ent).
• Direct evidence of ferrienterobactin (FeEnt) binding to FepB has been elusive due to high background binding.

Purpose of the Study:

• To provide direct evidence that FepB binds FeEnt.
• To investigate the utility of the LppOmpA membrane localization vehicle for studying periplasmic binding proteins.
• To confirm the classification of FeEnt transport within periplasmic permease systems.

Main Methods:

• Constructed plasmid pTX700 to express a fusion protein, LppOmpAFepB, in the E. coli outer membrane.
• Utilized outer membrane preparations and whole cells lacking the native receptor FepA for binding assays.
• Assessed FeEnt binding to outer membrane fractions and whole cells expressing the LppOmpAFepB fusion protein.

Main Results:

• Outer membrane expressing LppOmpAFepB showed significantly higher FeEnt binding compared to controls.
• Whole cells expressing the fusion protein also exhibited FeEnt binding, unaffected by energy poisons.
• The LppOmpA vehicle facilitated the localization of FepB to the outer membrane for functional analysis.

Conclusions:

• FepB directly binds FeEnt, confirming its role in the periplasmic permease category of iron transport.
• The LppOmpA system is a viable tool for localizing periplasmic binding proteins to the outer membrane for biochemical studies.
• This research clarifies the mechanism of iron-siderophore uptake in Gram-negative bacteria.