Functional characterization and transcriptional repression by Lacticaseibacillus paracasei DinJ-YafQ

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Genetics

Background:

• Bacterial type II toxin-antitoxin (TA) systems, like DinJ-YafQ, regulate essential cellular processes.
• While extensively studied in E. coli, orthologous systems in other bacteria remain less understood.

Purpose of the Study:

• To functionally characterize the DinJ-YafQ TA system from Lacticaseibacillus paracasei.
• To elucidate the molecular mechanisms of YafQ toxin activity and DinJ antitoxin neutralization.
• To investigate the DNA-binding and regulatory roles of DinJ and YafQ in L. paracasei.

Main Methods:

• Recombinant protein expression and purification of DinJ and YafQ from L. paracasei.
• In vitro enzymatic assays to assess YafQ RNase activity and DinJ neutralization.
• Electrophoretic mobility shift assays (EMSAs) and atomic force microscopy (AFM) to study DNA-protein interactions.
• In vivo reporter gene assays to confirm promoter regulation.

Main Results:

• Two L. paracasei YafQ orthologs exhibit distinct ribosomal RNA cleavage efficiencies, both neutralized by DinJ.
• DinJ and DinJ-YafQ complexes bind cooperatively to an inverted repeat sequence upstream of the TA operon promoter.
• The R13A mutation in DinJ's DNA-binding motif abolishes DNA binding for both DinJ and DinJ-YafQ.
• In vivo studies confirm promoter repression and reveal toxicity associated with high GFP reporter gene expression.

Conclusions:

• DinJ effectively neutralizes YafQ's toxic RNase activity in L. paracasei.
• DinJ and DinJ-YafQ act as transcriptional repressors by binding to their own promoter region.
• The DNA-binding domain of DinJ is crucial for the autoregulation of the type II TA system.