CC3 - Energy transduction through the gram negative cell envelope

What are the two major structural differences between Gram-positive and Gram-negative bacterial envelopes?

Gram-positive: thick peptidoglycan, single membrane. Gram-negative: thin peptidoglycan, inner membrane + outer membrane containing LPS.

What is the main structural component of the bacterial inner membrane?

Phospholipid bilayer.

What is the main structural component of the bacterial cell wall?

Peptidoglycan (PG).

What unique structural feature is present in Gram-negative but not Gram-positive bacteria?

An outer membrane containing lipopolysaccharide (LPS).

What forms the outer leaflet of the Gram-negative outer membrane?

Lipopolysaccharide (LPS).

Why does the Gram-negative outer membrane increase antibiotic resistance?

It acts as an additional permeability barrier that limits antibiotic entry.

Is the Gram-negative outer membrane energized by ATP or PMF?

No — the outer membrane lacks ATP and proton motive force.

Why must outer membrane processes be powered from the inner membrane?

The periplasm and outer membrane lack ATP and PMF, so energy must be transduced from the energized inner membrane.

What are the two primary energy sources used across the bacterial envelope?

Proton motive force (PMF) and ATP hydrolysis.

Which energy source powers bacterial flagellar rotation in many bacteria?

Proton motive force (PMF).

Which ion gradient can power gliding motility in some bacteria?

Na+ gradient.

Which energy source powers Type VI secretion systems?

ATP hydrolysis.

Which energy source drives LPS transport to the outer membrane?

ATP hydrolysis.

Which energy source drives TonB-dependent nutrient transport?

Proton motive force (PMF).

Name four substrates transported by TonB-dependent transporters.

Fe3+-siderophores, haem, vitamin B12, complex carbohydrates.

What is the function of the MotAB complex?

Acts as a PMF-driven stator that powers flagellar rotation.

What structural symmetry underpins stator rotary mechanisms such as MotA/MotB or ExbB/ExbD?

5:2 asymmetry (pentameric ring with dimer inside).

What conserved residue in ExbD/MotB is critical for proton translocation?

A conserved Asp residue in the transmembrane helix.

What model explains proton-driven rotation in MotA/MotB-like systems?

The alternating charge model.

In the alternating charge model, what drives rotation?

Cycles of protonation and deprotonation of a conserved Asp residue.

What is the function of the TonB system?

Transduces PMF from the inner membrane to power nutrient uptake through outer membrane TonB-dependent transporters.

Which inner membrane proteins form the stator complex for the TonB system?

ExbB (pentamer) and ExbD (dimer).

What is the role of TonB in nutrient transport?

TonB physically pulls on the plug domain of TonB-dependent transporters to open the pore.

What structural feature blocks the pore of TonB-dependent transporters?

A plug domain inside the β-barrel.

How does substrate binding affect TonB-dependent transporters?

It primes the transporter by inducing conformational changes that allow TonB engagement.

What is the TonB box?

An N-terminal region of TonB-dependent transporters that interacts with TonB.

How does TonB open the transporter pore?

By pulling on the TonB box and disrupting the plug domain.

Give two examples of TonB-dependent transporters in E. coli.

FhuA and BtuB.

What substrate does FhuA transport?

Ferrichrome (a siderophore).

What substrate does BtuB transport?

Vitamin B12.

Why is transport through TonB-dependent transporters considered active?

It requires energy transduction from the proton motive force.

What is the primary function of the Tol-Pal system?

Stabilization of the outer membrane during cell division.

What phenotype results from deletion of Tol-Pal components?

Outer membrane destabilization and cell division defects.

Which outer membrane lipoprotein is mobilized by the Tol-Pal system?

Pal.

What does Pal bind to in the envelope?

The outer membrane and peptidoglycan.

Is Pal permanently bound to peptidoglycan?

No — it can bind and unbind.

How does TolB affect Pal binding?

TolB binds Pal and releases it from peptidoglycan.

What is required to actively accumulate Pal at the division site?

TolQRA complex, TolB, and proton motive force.

How is Pal mobility different at the septum compared to elsewhere?

Pal is immobilized at the septum (PG-bound) and more mobile away from it.

What experimental technique measures Pal mobility in cells?

Fluorescence recovery after photobleaching (FRAP).

What does fluorescence recovery after photobleaching indicate?

Diffusion and mobility of fluorescently labeled proteins.

What happens to Pal accumulation at the septum in Tol-Pal mutants?

It is abolished.

How does TolA interact with TolB?

Through β-strand augmentation forming a force-resistant parallel β-sheet.

What mechanical role does TolA play in the Tol-Pal system?

TolA pulls TolB away from Pal using PMF-derived force.

What is the proposed mechanical cycle of Pal recruitment?

TolB releases Pal from PG → TolB-Pal diffuses → TolA binds TolB and strips it from Pal → Pal rebinds PG at septum.

What is the role of TolQ and TolR?

They form the PMF-driven stator complex in the inner membrane.

What is structurally similar between the TonB and TolA systems?

Both use PMF-driven stators and β-strand augmentation to transmit force.

Why is energy transduction necessary for outer membrane invagination during division?

The outer membrane is mechanically rigid and requires force to constrict.

What two additional proteins stabilize the outer membrane besides Pal?

Lpp and OmpA.

How many ATP molecules are hydrolyzed during LPS transport across the inner membrane?

Two ATP molecules.

Which protein complex inserts LPS into the outer membrane?

LptDE.

How is LPS transport across the envelope often described mechanistically?

Like a PEZ dispenser pushing LPS molecules outward.

Where is LPS ultimately deposited in the outer membrane?

In the outer leaflet via a lateral gate in LptDE.

Why is the outer membrane considered energy deficient?

It lacks ATP and proton motive force.

What general strategy allows inner membrane motors to power outer membrane processes?

Energy transduction via PMF-driven stator complexes that span the periplasm.