A Genetic Tool to Quantify trans-Translation Activity in Vivo

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Trans-translation is a crucial bacterial quality control mechanism that rescues stalled ribosomes.
• This process is essential for bacterial viability and the virulence of many pathogens.
• Understanding trans-translation is key to developing novel antibacterial strategies.

Purpose of the Study:

• To develop a reliable in vivo reporter system for quantifying bacterial trans-translation and associated proteolysis.
• To validate the reporter system using known genetic mutants.
• To identify and characterize novel inhibitors of trans-translation.

Main Methods:

• Development of a double-fluorescence reporter system for simultaneous measurement of trans-translation and proteolysis in vivo.
• Validation of the assay using bacterial mutants deficient in tmRNA, SmpB, and ClpP.
• Testing of potential inhibitors, including antisense tmRNA-binding RNA, an SmpB peptide mimic, and the oxadiazole derivative KKL-35.

Main Results:

• The double-fluorescence reporter system reliably quantified trans-translation and proteolysis activities.
• Antisense tmRNA-binding RNA and an SmpB peptide mimic were confirmed as potent in vivo inhibitors of trans-translation.
• The compound KKL-35 demonstrated inhibitory effects, but the study revealed it targets pathways beyond trans-translation.

Conclusions:

• The developed double-fluorescence reporter is a valuable tool for studying bacterial trans-translation and proteolysis in vivo.
• Novel inhibitors of trans-translation were identified, offering potential for antibacterial drug development.
• The findings highlight the complex mechanisms of action for compounds like KKL-35, necessitating further investigation into their broader cellular targets.