Two-component bacterial multidrug transporter, EbrAB: Mutations making each component solely functional.

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• The small multidrug resistance (SMR) family comprises multidrug efflux pumps.
• EmrE from Escherichia coli is a representative SMR protein functioning as a homo-oligomer.
• EbrAB from Bacillus subtilis is a novel SMR efflux pump requiring a hetero-oligomeric structure of EbrA and EbrB.

Purpose of the Study:

• To investigate the structural requirements for the hetero-oligomerization of the EbrAB efflux pump.
• To determine why individual EbrA and EbrB polypeptides do not confer multidrug resistance alone.
• To elucidate the role of hydrophilic loops and C-terminus regions in EbrAB function.

Main Methods:

• Site-directed mutagenesis of EbrA and EbrB.
• Analysis of multidrug resistance conferred by wild-type and mutant proteins.
• Expression studies of individual and combined polypeptides.

Main Results:

• Single expression of EbrA or EbrB does not confer multidrug resistance.
• Mutants of EbrA and EbrB lacking hydrophilic loops and C-terminus regions gained individual multidrug-resistance activity.
• The hydrophilic loops and C-terminus regions are crucial for the hetero-oligomeric assembly and function of EbrAB.

Conclusions:

• The EbrAB efflux pump necessitates a hetero-oligomeric configuration for its function.
• Specific regions (hydrophilic loops and C-terminus) in EbrA and EbrB are inhibitory for individual activity but essential for hetero-oligomer formation.
• These regions likely stabilize the distinct conformations required for the functional EbrAB complex.