Chloride Ions Are Required for Thermosipho africanus MurJ Function

Area of Science:

• Bacteriology
• Structural Biology
• Biochemistry

Background:

• Bacteria utilize the MurJ flippase to transport peptidoglycan precursor Lipid II across the cytoplasmic membrane, a crucial step for cell wall synthesis.
• MurJ proteins employ an alternating-access mechanism for Lipid II translocation, but the energy-coupling mechanism and role of ions remain unclear.
• Crystal structures suggest potential ion-binding sites in MurJ, but their functional significance in transport is unknown.

Purpose of the Study:

• To investigate the role of specific ions, particularly chloride and sodium, in the in vivo function of MurJ flippase from Thermosipho africanus (MurJ_Ta) and Escherichia coli (MurJ_Ec).
• To elucidate the ion requirements for MurJ-mediated Lipid II transport and understand potential differences in MurJ function across bacterial species.

Main Methods:

• In vivo functional assays were performed to assess the activity of MurJ_Ta and MurJ_Ec under varying ionic conditions.
• Site-directed mutagenesis was used to probe the function of residues within the putative sodium-binding site of MurJ_Ta.
• Comparative analysis of MurJ function in different bacterial species was conducted.

Main Results:

• Chloride ions were found to be essential for MurJ_Ta function, while sodium ions were not required.
• MurJ_Ec function was independent of both chloride and sodium ions.
• Mutations at the identified sodium-binding site in MurJ_Ta affected protein stability and/or function but did not abolish complementation, suggesting a non-essential role for this specific site in all contexts.

Conclusions:

• Chloride ions are necessary for the conformational changes that reset MurJ_Ta after Lipid II translocation, highlighting a specific ion requirement for this ortholog.
• MurJ orthologs exhibit functional diversity, with differing requirements for counterions, suggesting that while the general transport mechanism is conserved, specific mechanistic details vary across bacteria.
• Understanding these differences in MurJ ion dependency is crucial for developing MurJ as a broadly effective antibiotic target.