Death of the TonB Shuttle Hypothesis.

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• The ExbB-ExbD-TonB complex energizes active transport across the outer membrane.
• TonB's mechanism, whether it shuttles or remains membrane-bound, is debated.
• Previous studies yielded conflicting evidence regarding TonB's localization during energy transduction.

Purpose of the Study:

• To definitively determine if TonB shuttles during energy transduction.
• To resolve the conflicting models of TonB function.

Main Methods:

• Construction and analysis of a proteolytically stable ToxR-TonB fusion protein.
• Assessing TonB localization using proteinase K resistance and formaldehyde crosslinking.
• Re-evaluating previous experiments using a fluorescent probe.

Main Results:

• The ToxR-TonB fusion protein remained localized to the cytoplasmic membrane.
• TonB's conformation and interaction with ExbD were dependent on proton motive force.
• Previous evidence for TonB shuttling was based on a permeable probe, not actual shuttling.

Conclusions:

• TonB does not shuttle; it remains anchored to the cytoplasmic membrane.
• The proton motive force drives TonB-mediated transport while TonB is membrane-bound.
• Reinterpretation of prior data suggests a signal transduction pathway from the outer membrane to the cytoplasm.