Inner membrane components of the plasmid pKM101 type IV secretion system TraE and TraD are DNA-binding proteins

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Antimicrobial resistance (AMR) is a major global health threat.
• Plasmid transfer via bacterial conjugation, mediated by type IV secretion systems (T4SS), drives AMR spread.
• Previous studies suggest T4SS components interact with DNA during conjugation.

Purpose of the Study:

• To investigate the DNA-binding properties of specific T4SS inner membrane proteins, TraD and TraE, from Escherichia coli pKM101.
• To elucidate the mechanism of action of the conjugation inhibitor BAR-072.
• To identify potential targets for novel antimicrobial resistance inhibitor development.

Main Methods:

• Purification of TraD and TraE proteins from Escherichia coli.
• Electrophoretic mobility shift assays (EMSA) to assess DNA binding.
• Fluorescence polarization to quantify DNA-binding affinity.
• Site-directed mutagenesis to identify key amino acid residues.

Main Results:

• Both purified TraD and TraE proteins bind single-stranded and double-stranded DNA with nanomolar affinity.
• The conjugation inhibitor BAR-072 was shown to inhibit TraE DNA binding in vitro.
• Mutagenesis identified conserved amino acids essential for conjugation, likely involved in DNA interaction.

Conclusions:

• TraD and TraE are DNA-binding proteins crucial for the function of the pKM101 T4SS.
• BAR-072 likely functions by interfering with TraE's DNA binding activity.
• Conserved residues in TraD and TraE represent potential targets for developing new inhibitors to combat antimicrobial resistance.